Mouse Embryonic Fibroblast Cells Research Articles

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1

Reactive oxygen species (ROS) are highly reactive and its accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress.Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-β-activated kinase (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contribute to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.

Physicochemical and Biological Characterization of Propolis-Loaded Composite Polyamide-6/Soybean Protein Nanofibers for Wound Healing Applications

Dressings are pivotal in aiding wound recovery, yet they are burdened by significant constraints. In addition to possessing antibacterial properties, the effectiveness of a wound dressing must be rigorously assessed for its potential utility across diverse medical applications. Propolis (Pro) is widely acknowledged for its antioxidant, antibacterial, and anti-inflammatory properties, positioning it as a promising candidate for wound healing and tissue engineering applications. This study presents a novel composite nanofiber (NF) material comprising polyamide-6 (PA6) and soybean protein as matrix components, incorporating Pro as a payload. We aimed to mimic the extracellular matrix, thus promotion the proliferation and adhesion of mouse embryonic fibroblast cells (NIH3T3) on the scaffold's surface. The morphology and composition of the NFs, examined through scanning electron microscopy (SEM) imaging and Fourier-transform infrared (FTIR) spectroscopy. The Pro-loaded fibers exhibited diameters ranging from 102 to 270nm. Notably, the NFs demonstrated a dose-dependent increase in porosity, water vapor transmission rate, and fluid uptake capacity. However, the hydrophilic categorized nanofibers (NFs) did not exhibit a concentration-dependent effect on contact angle measurements. After a 15-day period, the Pro-loaded fibers experienced degradation within the range of 39-53%. Using UV-visible spectrophotometry, the amount of pro released was measured. The rates of release were found to be 29.93%, 41.02%, and 60.06% within 24hours for PA6/Soy/Pro5%, PA6/Soy/Pro10%, and PA6/Soy/Pro20% NFs, respectively. Importantly, our study showcased the significant inhibitory effects of Pro-loaded fibers on the growth of E. coli and S. aureus, with inhibition increasing proportionally with higher Pro concentrations. This observation underscores the potential of Pro-based composite NFs as effective antimicrobial agents for wound dressings and tissue scaffolds. Furthermore, the biocompatibility of the fabricated nanofibrous mats was confirmed through the MTT test, affirming the non-toxic nature of all formulations. SEM imaging further demonstrated the optimal adhesion of cells on the NFs, confirming the findings of the MTT test and highlighting the suitability of Pro-loaded NFs for cell attachment and proliferation.

The Metabolic and Lipidomic Fingerprint of Torin1 Exposure in Mouse Embryonic Fibroblasts Using Untargeted Metabolomics

Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography—high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5′-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators.

Synthesis and evaluation of tryptanthrin derivatives as promising anticancer agents: In vitro, in silico, and SAR insights

In this study, with the aim of identifying new anticancer agents, we synthesized a series of tryptanthrin derivatives. All the synthesized compounds were investigated for their anticancer potential in-vitro, using an MTT-based assay against a panel of three different cancer cell lines (A-549, MCF-7, and MDA-MB-231). Amongst the synthesized derivatives, compound 3 showed excellent anticancer activity, with GI50 values of 5.03 ± 0.77 µM, 3.76 ± 0.55 µM, and 4.20 ± 0.50 µM against A-549, MDA-MB-231 and MCF-7 cells, respectively. Furthermore, these molecules did not show any cytotoxic effect against normal mouse embryonic fibroblast (NIH/3T3) cell lines suggesting no intrinsic cytotoxicity. In this study, ADME and toxicity profiles of the synthesized compounds were also predicted using in silico techniques. According to the findings, all of the synthesized derivatives are nontoxic and non-carcinogenic in the biological system and showed good ADME properties. Finally, potent compounds were subjected to in silico molecular docking and simulation studies were carried out against maternal embryonic leucine zipper kinase (MELK) to uncover potential protein-ligand interactions and affinity at the binding site. This study will help in the future development of novel anticancer agents.

Multifunctional 58S Bioactive Glass/Silver/Cerium Oxide-Based Biocomposites with Effective Antibacterial, Cytocompatibility, and Mechanical Properties.

58S bioactive glass (BG) has effective biocompatibility and bioresorbable properties for bone tissue engineering; however, it has limitations regarding antibacterial, antioxidant, and mechanical properties. Therefore, we have developed BGAC biocomposites by reinforcing 58S BG with silver and ceria nanoparticles, which showed effective bactericidal properties by forming inhibited zones of 2.13 mm (against Escherichia coli) and 1.96 mm (against Staphylococcus aureus; evidenced by disc diffusion assay) and an increment in the antioxidant properties by 39.9%. Moreover, the elastic modulus, hardness, and fracture toughness were observed to be increased by ∼84.7% (∼51.9 GPa), ∼54.5% (∼3.4 GPa), and ∼160% (∼1.3 MPam1/2), whereas the specific wear rate was decreased by ∼55.2% (∼1.9 × 10-11 m3/Nm). X-ray diffraction, high-resolution transmission electron microscopy, and field emission scanning electron microscopy confirmed the fabrication of biocomposites and the uniform distribution of the nanomaterials in the BG matrix. The addition of silver nanoparticles in the 58S BG matrix (in BGA) increased mechanical properties by composite strengthening and bactericidal properties by damaging the cytoplasmic membrane of bacterial cells. The addition of nanoceria in 58S BG (BGC) increased the antioxidant properties by 44.5% (as evidenced by the 2,2-diphenyl-1-picrylhydrazyl assay). The resazurin reduction assay and MTT assay confirmed the effective cytocompatibility for BGAC biocomposites against mouse embryonic fibroblast cells (NIH3T3) and mouse bone marrow stromal cells. Overall, BGAC resulted in mechanical properties comparable to those of cancellous bone, and its effective antibacterial and cytocompatibility properties make it a good candidate for bone healing.

Methylene blue treatment with zero-valent iron/pyrite/H2O2 system under static and continuous flow conditions: Reaction mechanism and toxicity evaluation

Laboratory batch and continuous flow studies, coupled with surface and toxicity analysis, were performed to evaluate methylene blue treatment by heterogeneous Fenton process using zero-valent iron (ZVI) and pyrite as co-catalyst. The use of pyrite in batch reactors significantly enhanced methylene blue treatment by the ZVI/H2O2 system because of improved iron redox cycling. The continuous-flow experiments revealed that the reactor performance increased in the order of: ZVI/H2O2 < pyrite/H2O2 < ZVI/pyrite/H2O2 under dynamic flow conditions. The methylene blue treatment by the ternary ZVI/pyrite/H2O2 system was described by an initial degradation of methylene blue with *OH radicals, followed by the adsorption and/or co-precipitation of degradation intermediates with some spherical particles. The surface analysis showed that these spherical particles formed, even at pH less than 4. The genotoxicity and cytotoxicity tests performed on mouse embryonic fibroblast (3 T3-L1) and human embryonic kidney (HEK293) cell lines showed that the Fenton treatment of methylene blue using the ZVI/pyrite/H2O2 system resulted in the formation of degradation species with much lower toxicity levels relative to methylene blue. Moreover, the Fenton degradation species of methylene blue significantly enhanced the metabolic activity of several bacterial strains, including E. coli ATCC 8739 and P. aeruginosa PAO1.

Synthesis, Cytotoxic Activity Evaluation and Molecular Docking Studies of Some Benzimidazole Derivatives

In this study, the synthesis of 2-(2-acetyl-1H-benzimidazol-1-yl)-1-arylethanone (3a-3d) and 1-methyl-3-phenyl-benzo[4,5]imidazo[1,2-a]pyrazine derivatives (4a-4d) and to investigate their cytotoxic activity were aimed. APCI, IR, 1HNMR, and 13CNMR spectra were utilized to determine the structure of the synthesized compounds. The cytotoxic activity of selected compounds were detected in A549 (human lung carcinoma) and NIH3T3 (mouse embryonic fibroblasts) cell lines. Compounds 4c and 4d were found to be selectively cytotoxic against A549 and NIH3T3 cell lines. Molecular docking studies were performed using the data retrieved from the Protein Data Bank server (PDBID: 4QTX).

Faster structured illumination microscopy using complementary encoding-based compressive imaging

Structured illumination microscopy (SIM) has been widely applied to investigate intricate biological dynamics due to its outstanding super-resolution imaging speed. Incorporating compressive sensing into SIM brings the possibility to further improve the super-resolution imaging speed. Nevertheless, the recovery of the super-resolution information from the compressed measurement remains challenging in experiments. Here, we report structured illumination microscopy with complementary encoding-based compressive imaging (CECI-SIM) to realize faster super-resolution imaging. Compared to the nine measurements to obtain a super-resolution image in a conventional SIM, CECI-SIM can achieve a super-resolution image by three measurements; therefore, a threefold improvement in the imaging speed can be achieved. This faster imaging ability in CECI-SIM is experimentally verified by observing tubulin and actin in mouse embryonic fibroblast cells. This work provides a feasible solution for high-speed super-resolution imaging, which would bring significant applications in biomedical research.

Evaluation of anticancer effect of Cladophora glomerata algae extract

Cladophora glomerata has numerous biologically active properties and is considered one of the most essential medicinal algae. The purpose of this research was to investigate the anticancer efficacy of Cladophora glomerata algae extract against human hepatocellular carcinoma (HepG2), human cervical carcinoma (HeLa), and normal mouse embryonic fibroblast (MEF) cell lines. The needed algae was found in the Hassan Al-Hamoud River , Baquba, Iraq. The chemical detection of some chemical components of the ethanolic extract of C. glomerata revealed that the extract contains a group of active compounds. The study showed significant variation (p&lt;0.05) among inhibition percentages of cancer cell line HepG2, Hella, and MEF cell lines that were treated with different concentrations (15.1, 31.2, 62.5, 125, 250, 500, and 1000) μg/ml of C. glomerata extract. The outcomes showed that increased inhibition percentages of the above cell line were associated with increased concentrations. The inhibition percentage of HepG2, Hella, and  MEF cell lines were  1.6 ± 30.88, 1.7 ± 14.10, and 1.2 ± 2.31 at concentration 15.1 (mg/ml), and was 3.8 ± 84.90, 3.6 ± 88.29, and 3.9 ± 23.2, respectively at concentration 1000 (mg/ml). The study concluded that the C. glomerata extract with different concentrations significantly inhibited cancer cell lines (HepG2 and Hela) and ME because they have antiproliferative and antioxidant activity against cancers. The extract's inhibitory impact begins at low doses and increases with increasing concentration. The study would be beneficial to use macroalgae as new and sustainable sources of bioactive compounds against cancer.  

Exploring the Role of Inulin in Targeting the Gut Microbiota: An Innovative Strategy for Alleviating Colonic Fibrosis Induced By Irradiation.

The use of radiation therapy to treat pelvic and abdominal cancers can lead to the development of either acute or chronic radiation enteropathy. Radiation-induced chronic colonic fibrosis is a common gastrointestinal disorder resulting from the above radiation therapy. In this study, we establish the efficacy of inulin supplements in safeguarding against colonic fibrosis caused by irradiation therapy. Studies have demonstrated that inulin supplements enhance the proliferation of bacteria responsible to produce short-chain fatty acids (SCFAs) and elevate the levels of SCFAs in feces. In a mouse model of chronic radiation enteropathy, the transplantation of gut microbiota and its metabolites from feces of inulin-treated mice were found to reduce colonic fibrosis in validation experiments. Administering inulin-derived metabolites from gut microbiota led to a notable decrease in the expression of genes linked to fibrosis and collagen production in mouse embryonic fibroblast cell line NIH/3T3. In the cell line, inulin-derived metabolites also suppressed the expression of genes linked to the extracellular matrix synthesis pathway. The results indicate a novel and practical approach to safeguarding against chronic radiation-induced colonic fibrosis.

Carboxymethylated and Sulfated Furcellaran from Furcellaria lumbricalis and Its Immobilization on PLA Scaffolds.

This study involved the creation of highly porous PLA scaffolds through the porogen/leaching method, utilizing polyethylene glycol as a porogen with a 75% mass ratio. The outcome achieved a highly interconnected porous structure with a thickness of 25 μm. To activate the scaffold's surface and improve its hydrophilicity, radiofrequency (RF) air plasma treatment was employed. Subsequently, furcellaran subjected to sulfation or carboxymethylation was deposited onto the RF plasma treated surfaces with the intention of improving bioactivity. Surface roughness and water wettability experienced enhancement following the surface modification. The incorporation of sulfate/carboxymethyl group (DS = 0.8; 0.3, respectively) is confirmed by elemental analysis and FT-IR. Successful functionalization of PLA scaffolds was validated by SEM and XPS analysis, showing changes in topography and increases in characteristic elements (N, S, Na) for sulfated (SF) and carboxymethylated (CMF). Cytocompatibility was evaluated by using mouse embryonic fibroblast cells (NIH/3T3).

High efficacy fluconazole loaded ZnO‐poly (vinyl alcohol) nanocomposite: Interpretive breakpoints for biological applications

AbstractNanotechnology, a versatile field, holds promise in diverse applications, such as advanced pharmaceutical techniques and innovative chemical compound fabrication. Recently, the World Health Organization (WHO) has identified sepsis as a global health priority, attributing most sepsis‐related deaths to the underlying infection. Sepsis is a complex disease that manifests in various ways, depending on factors, such as pathogen involved, mode of transmission, and the patient's immune competence. This study focuses on synthesizing zinc oxide (ZnO) through an in‐situ precipitation method and employing a solution‐based technique to coat the inorganic ZnO nanomaterial with the antimicrobial drug fluconazole (FLZ), resulting in a FLZ‐ZnO composite. Further enhancement is achieved by modifying the composite with poly(vinyl alcohol) (PVA) to improve mechanical strength, physicochemical characteristics, and the interfacial network between ZnO and FLZ. Characterization through X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), particle size distribution (PSD), and thermo gravimetric analysis (TGA) confirms the synthesized compounds are stoichiometric in nature. The FLZ‐ZnO‐PVA nanocomposite demonstrates significant antifungal activity against C. albicans and A. niger, as well as enhanced antibacterial activity against E. coli and S. aureus evaluated through well diffusion technique. In vitro cellular compatibility assessment using the MTT assay with NIH‐3T3 cells reveals exceptional viability (above 75%) and negligible cytotoxicity at a concentration of 1.56 μg/mL, indicating high biosafety. The FLZ‐ZnO‐PVA nanocomposite exhibits outstanding biological performance, making it a promising candidate for clinical applications in preventing sepsis and prospective infections.Highlights Developments and preparation of FLZ‐ZnO‐PVA nanocomposite. FLZ‐ZnO‐PVA nanocomposite shows optimum antimicrobial activity. FLZ‐ZnO‐PVA shows cytotoxicity against the mouse embryonic fibroblast cell line. FLZ‐ZnO‐PVA could be used as a suitable material for treatment of sepsis.

Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

Zinc complexes of chloroquine and hydroxychloroquine versus the mixtures of their components: Structures, solution equilibria/speciation and cellular zinc uptake

The zinc complexes of chloroquine (CQ; [Zn(CQH+)Cl3]) and hydroxychloroquine (HO-CQ; [Zn(HO-CQH+)Cl3]) were synthesized and characterized by X-Ray structure analysis, FT-IR, NMR, UV–Vis spectroscopy, and cryo-spray mass spectrometry in solid state as well as in aqueous and organic solvent solutions, respectively. In acetonitrile, up to two Zn2+ ions bind to CQ and HO-CQ through the tertiary amine and aromatic nitrogen atoms (KN-aminCQ = (3.8 ± 0.5) x 104 M−1 and KN-aromCQ = (9.0 ± 0.7) x 103 M−1 for CQ, and KN-aminHO-CQ = (3.3 ± 0.4) x 104 M−1 and KN-aromHO-CQ = (1.6 ± 0.2) x 103 M−1 for HO-CQ). In MOPS buffer (pH 7.4) the coordination proceeds through the partially deprotonated aromatic nitrogen, with the corresponding equilibrium constants of KN-arom(aq)CQ = (3.9 ± 1.9) x 103 M−1and KN-arom(aq)HO-CQ = (0.7 + 0.4) x 103 M−1 for CQ and HO-CQ, respectively. An apparent partition coefficient of 0.22 was found for [Zn(CQH+)Cl3]. Mouse embryonic fibroblast (MEF) cells were treated with pre-synthesized [Zn((HO-)CQH+)Cl3] complexes and corresponding ZnCl2/(HO-)CQ mixtures and zinc uptake was determined by application of the fluorescence probe and ICP-OES measurements. Administration of pre-synthesized complexes led to higher total zinc levels than those obtained upon administration of the related zinc/(hydroxy)chloroquine mixtures. The differences in the zinc uptake between these two types of formulations were discussed in terms of different speciation and character of the complexes. The obtained results suggest that intact zinc complexes may exhibit biological effects distinct from that of the related zinc/ligand mixtures.

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

Exploring marine algae-derived phycocyanin nanoparticles as a safe and effective adjuvant for sunscreen systems

BackgroundUV radiation (UV) exposure risks skin damage and cancer due to DNA damage and oxidative stress. Synthetic chemical sunscreens that protect against UV radiation can have health and environmental concerns. This study explores phycocyanin (PC), a marine algae-derived natural photoprotective compound, and its crosslinked nanoparticles (PCNP) as safe and effective adjuvants for sunscreen systems.MethodsPCNP was synthesized via genipin-crosslinking. PC and PCNP biocompatibility were assessed on mouse embryonic fibroblast cells. ABTS evaluated antioxidant activity, and the UV absorption capacity of PC and PCNP were analyzed. PCNP skin permeability was tested in vitro and in vivo. Gel formulations with PCNP were examined for UV absorption effects.ResultsPCNP showed good biocompatibility, maintaining cell viability above 90% across concentrations. Both PC and PCNP demonstrated concentration-dependent antioxidant activity, efficiently scavenging free radicals. PCNP exhibited enhanced UV absorption in the UVB range compared to PC alone. Skin permeation studies displayed limited PCNP penetration through skin layers. In vivo, absorption assessments indicated PCNP localized mainly in the stratum corneum. PCNP-containing gels displayed improved UV absorption compared to gels without PCNP.ConclusionThis study showcases PCNP’s potential as a natural and safe adjuvant for sunscreen with enhanced UV protection capabilities. PCNP preserved antioxidant activity, displayed limited skin penetration, and enhanced UV absorption. The findings suggest PCNP’s promise as a viable alternative to synthetic sunscreen agents, delivering effective photoprotection while minimizing health and environmental concerns.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Slowly progressive cell death induced by GPx4-deficiency occurs via MEK1/ERK2 activation as a downstream signal after iron-independent lipid peroxidation.

Glutathione peroxidase 4 (GPx4) is an antioxidant enzyme that reduces phospholipid hydroperoxide. Studies have reported that the loss of GPx4 activity through anticancer drugs leads to ferroptosis, an iron-dependent lipid peroxidation-induced cell death. In this study, we established Tamoxifen-inducible GPx4-deficient Mouse embryonic fibroblast (MEF) cells (ETK1 cells) and found that Tamoxifen-inducible gene disruption of GPx4 induces slow cell death at ~72 h. In contrast, RSL3- or erastin-induced ferroptosis occurred quickly within 24 h. Therefore, we investigated the differences in these mechanisms between GPx4 gene disruption-induced cell death and RSL3- or erastin-induced ferroptosis. We found that GPx4-deficiency induced lipid peroxidation at 24 h in Tamoxifen-treated ETK1 cells, which was not suppressed by iron chelators, although lipid peroxidation in RSL3- or erastin-treated cells induced ferroptosis that was inhibited by iron chelators. We revealed that GPx4-deficient cell death was MEK1-dependent but RSL3- or erastin-induced ferroptosis was not, although MEK1/2 inhibitors suppressed both GPx4-deficient cell death and RSL3- or erastin-induced ferroptosis. In GPx4-deficient cell death, the phosphorylation of MEK1/2 and ERK2 was observed 39 h after lipid peroxidation, but ERK1 was not phosphorylated. Selective inhibitors of ERK2 inhibited GPx4-deficient cell death but not in RSL3- or erastin-induced cell death. These findings suggest that iron-independent lipid peroxidation due to GPx4 disruption induced cell death via the activation of MEK1/ERK2 as a downstream signal of lipid peroxidation in Tamoxifen-treated ETK1 cells. This indicates that GPx4 gene disruption induces slow cell death and involves a different pathway from RSL3- and erastin-induced ferroptosis in ETK1 cells.

Optimization of Deep Eutectic Solvents Enables Green and Efficient Cryopreservation.

Cryopreservation presents significant opportunities for biomedical applications including cell therapy, tissue engineering, and assisted reproduction. Dimethyl sulfoxide (DMSO), the most commonly used cryoprotectant (CPA), can be added to cells to prevent cryogenic damage. However, the toxicity of cryoprotectants restrains its further development in many areas with safety concerns such as clinical treatment. Therefore, the development of low-toxicity cryoprotectants is essential for medical research. This work reports deep eutectic solvents (DES) as naturally biocompatible osmoprotectants for green and efficient cryopreservation of human umbilical cord mesenchymal stem cells (HuMSC), which may be an ideal alternative to DMSO. The six types of DESs were explored for thermal properties, toxicity, and permeability in cells. Raman spectroscopy and viscosity studies showed that DES exhibited an improved hydrogen-bonding system as the temperature decreased. By optimizing the freezing process (cooling rate, incubation time, and loading procedure) of DES, the viability of mouse embryonic fibroblast cells (NIH-3T3) after thawing was significantly improved. The HuMSC were successfully preserved with no significant difference (p > 0.05) in cell viability (94.65%) after thawing compared with DMSO, which preserved the cell differentiation function and improved the cell proliferation rate. The mechanism of DES in cryopreservation was investigated, and it was found that DES could bind water molecules and effectively inhibit the growth of ice crystals during ice recrystallization, reducing mechanical damage to cells. This study highlights the excellent performance of DES as a low-toxicity CPA for stem cell preservation, which may be a significant advance for future clinical cell therapy.

Transcriptional regulation of FACT involves Coordination of chromatin accessibility and CTCF binding

Histone chaperone FACT is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair, and also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.