Mouse Embryonic Fibroblast Cell Line Research Articles

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).

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<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.

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.

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.

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.

Fabrication and evaluation of silver-doped magnesium oxide nanocomposite coatings for orthopaedics applications

Stainless steel 316L (SS 316L) is primarily used as implant material in orthopaedic applications for the treatment of bone fractures and joint replacement surgeries due to its excellent mechanical strength and cost-effectiveness. However, its long-term implication, susceptibility to infection and subsequent lack of integration with surrounding tissues remain significant challenges resulting in implant failure. To address these issues, the development of advanced coatings with improved antibacterial properties, biocompatibility, wear and corrosion resistance is of paramount importance. In this study, the potential of an Ag doped MgO nanocomposite coating on stainless steel 316L substrates has been explored for orthopaedic applications. The Ag-MgO nanocomposite was synthesised using the hydrothermal method. The composition, crystallinity, and morphology of the synthesised nanocomposite was investigated using different techniques such as X-ray diffraction (XRD), Fourier Transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) with an energy dispersive X-ray (EDX) attachment. SEM and XRD results confirmed, that the synthesised nanocomposite had a platelet shape with a cubical crystal lattice network and an average size of about 10nm. The spin coating technique was employed to produce an Ag-MgO nanocomposite coating onto the SS 316L substrate. The bactericidal efficiency of Ag-MgO nanocomposite-coated SS 316L substrates was examined against Staphylococcus aureus and Escherichia coli bacteria using well diffusion method, while an MTT assay was performed to determine the cytotoxicity of the uncoated and coated SS 316L substrate against mouse embryonic fibroblast cell line (NIH-3T3). The effectiveness of wear and friction resistance of Ag-MgO nanocomposite-coated substrate was also evaluated via a ball-on-disc tribometer. Furthermore, corrosion behaviour evaluation using a linear polarisation resistance method confirms the significant improvement in corrosion resistance of coated substrate than the uncoated substrate. Overall, the results demonstrated that the Ag-MgO nanocomposite coating on SS 316L substrates improves its performance for orthopaedic applications.

Inclusion phenomenon of β-carboline alkaloids with sulfonatocalix[4]arene: Photophysical, cytotoxicity and theoretical study

β-carbolines are nonpolar heterocyclic aromatic amines known as Harman and Norharman. Harman and Norharman, with anticancer effects, have recently attracted much attention but have experienced difficulty in their pharmacological development due to their low oral bioavailability. In this article, we describe the complexation of Harman (H) and Norharman (NH) with p-sulfonatocalix[4]arene (SCX4) as a feasible option to improve its solubility and, as a result, improve the cytotoxic effect on cell lines. Spectroscopic techniques, such as UV–visible absorption, fluorescence, FT-IR, FE-SEM, HRMS, NMR techniques, and molecular docking calculations, were used to analyze these inclusion complexes H ⊂ SCX4 and NH ⊂ SCX4. UV–visible absorption and steady-state fluorescence spectroscopy were used to investigate the interaction between Harman and Norharman with p-sulfonatocalix[4]arene (SCX4). A simple and precise HPLC-based approach has assessed the retention times of H, NH, SCX4, and its complexes. Additionally, an in vitro cytotoxicity study revealed that the synthesized complexes were not toxic to the mouse embryonic fibroblast (3 T3-L1) cell line. Therefore, the present study confirmed that the H ⊂ SCX4 and NH ⊂ SCX4 complexes are readily soluble in an aqueous medium that does not cause toxicity in the primary cell line and might provide a feasible option for increasing the solubility of these class of poorly soluble drugs.

In-vitro cytotoxic potential of aerial parts of Leutea avicennae Mozaff. in different Human cancer cell lines

Medicinal plants such as Leutea avicennae Mozaff. (Apiaceae) have been shown some biological potential for preventing and treating diseases. Fractions and isolated compounds were tested on colon carcinoma (HT-29), cervical carcinoma (HeLa), breast carcinoma (MCF-7), and mouse embryonic fibroblast (NIH/3T3) cell lines. The BSLT method was used for the assessment of the general toxicity of the petroleum ether (PET), chloroform (CHCl3), ethyl acetate (EtOAc), and methanol (MeOH) fractions obtained from the aerial parts of L. avicennae. 1H-NMR and 13 C-NMR spectroscopy were used for structure elucidation. Five compounds, including two coumarins, osthole and umbelliferone, a diterpene phytol, β-sitosterol, and lauric acid, were isolated for the first time from L. avicennae. Osthole showed potent cytotoxic activity against MCF-7 and HT-29 cell lines with IC50 values of 4.23 ± 0.26 and 12.11 ± 0.13 µg/mL, respectively. Phytol demonstrated potent cytotoxic activity towards MCF-7 and HeLa cell lines with IC50 values of 6.80 ± 0.08 and 12.27 ± 0.18 µg/mL, respectively.

AS1411aptamer conjugated liposomes for targeted delivery of arsenic trioxide in mouse xenograft model of melanoma cancer

Development of AS1411aptamer-conjugated liposomes for targeted delivery of arsenic trioxide is the primary goal of this study. AS1411aptamer was used as ligand to target nucleolin, which is highly expressed on the surface of melanoma cancer cells. The targeted liposomes were constructed by the thin film method, and arsenic trioxide was loaded as cobalt (II) hydrogen arsenite (CHA) to increase the loading efficiency and stability of the liposomes. The liposomal structure was characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and field emission scanning electron microscopy (FESEM). In addition, particle sizes and zeta potential of the CHA-loaded liposomes (CHAL) and aptamer-functionalized CHA-loaded liposomes (AP-CHAL) were determined. In vitro cytotoxicity of CHAL and AP-CHAL were evaluated using MTT assay in murine melanoma (B16) and mouse embryonic fibroblast (MEF) cell lines. The encapsulation efficiency of CHAL and AP-CHAL was reported as 60.2 ± 6.5% and 58.7 ± 4.2%, respectively. In vivo antitumor activity of CHAL and AP-CHAL in the B16 tumor-xenograft mouse model was dramatically observed. All mice of both groups survived until the end of treatment and showed body weight gain. The tumor protrusion completely disappeared in 50% of the mice in these groups. Furthermore, histopathology studies demonstrated that CHAL and AP-CHAL did not induce significant toxicity in healthy mice tissues. However, unlike the CHAL group, which showed an initial increase in tumor volume, a specific antitumor effect was observed in the AP-CHAL group from the beginning of treatment. The results showed that AP-CHAL can be used as an effective drug delivery system with high potential in the treatment of solid tumors.

Processing and biomedical applications of novel eco-sustainable fluconazole–loaded zinc oxide (ZnO) encapsulated chitosan (CS) biopolymer nanocomposite by inhibiting microbe species against candidiasis

Drug resistance and a lack of effective antifungal treatments have spurred the research and development of novel therapeutics to combat the fungus Candida albicans. Candida is ubiquitous yeast that is often colonized on the skin and mucosal membranes and is implicated in opportunistic fungal disorders all over the globe. In particular, nanoparticles (NPs) made of metals and metal oxides are a novel kind of functional nanohybrids with fascinating physio-chemical characteristics that have potential uses in nanoscience and medicine. In this current novel research attempt, a decisive biocompatible nanocomposite material with conflation of organic and inorganic sources has been successfully developed using a bis-triazole antifungal agent, fluconazol (FCZ)-assimilated zinc oxide (ZnO) as filler, and chitosan (CS) as matrix by a solution-based chemical method for potent biomedical applications. The eco-sustainable biomaterial ZnO NPs has been synthesized with a simple in situ precipitation method, in which the potent antifungal drug FCZ was implemented into the system to improve its strength, and the system was further modified by employing CS biopolymer. CS is an amorphous, translucent organic biopolymer with a structure unit similar to the polysaccharide structure of the extracellular matrix. This may provide mechanical strength to the nanocomposite by creating interfacial bonding between them. Such a novel nanocomposite has been physicochemically characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Ultra violet (UV), Scanning electron microscope (SEM), and particle size distribution (PSD). Additionally, the well diffusion technique was used to evaluate the ZnO-NPs and to develop FCZ-ZnO-CS nanocomposite for antibacterial and antifungal activities against Escherichia coli, Staphylococcus aureus, Candida albicans, and A. niger. The FCZ-ZnO-CS functionalized nanocomposite has excellent antifungal activity against Candida albicans and Aspergillus niger and optimum antibacterial activity against Escherichia coli and Staphylococcus aureus. Cytotoxicity studies demonstrated that the developed nanocomposite material FCZ-ZnO-CS has significant cytotoxicity against the mouse embryonic fibroblast (NIH-3T3) cell line. These results reveal that ZnO-incorporated fluconazole-chitosan (FCZ-ZnO-CS) could be used as an ideal material for candidiasis applications.

Melt Polycondensation Strategy for Amide-Functionalized l-Aspartic Acid Amphiphilic Polyester Nano-assemblies and Enzyme-Responsive Drug Delivery in Cancer Cells.

Aliphatic polyesters are intrinsically enzymatic-biodegradable, and there is ever-increasing demand for safe and smart next-generation biomaterials including drug delivery nano-vectors in cancer research. Using bioresource-based biodegradable polyesters is one of the elegant strategies to meet this requirement; here, we report an l-amino acid-based amide-functionalized polyester platform and explore their lysosomal enzymatic biodegradation aspects to administrate anticancer drugs in cancer cells. l-Aspartic acid was chosen and different amide-side chain-functionalized di-ester monomers were tailor-made having aromatic, aliphatic, and bio-source pendant units. Under solvent-free melt polycondensation methodology; these monomers underwent polymerization to yield high molecular weight polyesters with tunable thermal properties. PEGylated l-aspartic monomer was designed to make thermo-responsive amphiphilic polyesters. This amphiphilic polyester was self-assembled into a 140 ± 10 nm-sized spherical nanoparticle in aqueous medium, which exhibited lower critical solution temperature at 40-42 °C. The polyester nano-assemblies showed excellent encapsulation capabilities for anticancer drug doxorubicin (DOX), anti-inflammatory drug curcumin, biomarkers such as rose bengal (RB), and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt. The amphiphilic polyester NP was found to be very stable under extracellular conditions and underwent degradation upon exposure to horse liver esterase enzyme in phosphate-buffered saline at 37 °C to release 90% of the loaded cargoes. Cytotoxicity studies in breast cancer MCF 7 and wild-type mouse embryonic fibroblasts cell lines revealed that the amphiphilic polyester was non-toxic to cell lines up to 100 μg/mL, while their drug-loaded polyester nanoparticles were able to inhibit the cancerous cell growth. Temperature-dependent cellular uptake studies further confirmed the energy-dependent endocytosis of polymer NPs across the cellular membranes. Confocal laser scanning microscopy assisted time-dependent cellular uptake analysis directly evident for the endocytosis of DOX loaded polymer NP and their internalization for biodegradation. In a nutshell, the present investigation opens up an avenue for the l-amino acid-based biodegradable polyesters from l-aspartic acids, and the proof of concept is demonstrated for drug delivery in the cancer cell line.

Abstract A028: An isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clonal parent

Abstract Shortly after the formation of the NCI Ras Initiative, a H/N/KRAS-less mouse embryonic fibroblast (MEF) cell line panel was generated, validated, and shared internationally to aid the research community in its reinvigorated effort to apply new technical advancements to understanding the structure and function of Ras and to ultimately design inhibitors. While this panel has been beneficial to the Ras research community, an inherent variability due in part to the ploidy of the parental pool from which the panel was derived complicated comparisons between isolates and alleles. We have minimized this variation by utilizing a diploid clone derived from the DU1473 MEF; Hras-/-; Nras-/-; Kraslox/lox; RERTert/ert - parental cell line. A diploid cell population was sorted, cloned and clones were cultured continuously for up to 20 passages to identify those that retained a diploid signature. These clones were further screened for p53 mutations and mouse exome sequenced from which a final clone was selected to develop the improved cell line panel. The selected clone was treated with 4-hydroxytamoxifen (4-OHT) to activate the Cre-recombinase which excised a portion of the endogenous Kras located between two loxP sites effectively arresting cells in a senescent G1 state. Molecular verification of Kras removal was performed, and the cells were then transduced with a third generation VSV-G pseudotyped lentiviral vector which delivered different versions of the human Ras transgene as well as an antibiotic selection marker. This transduction restored the proliferative ability of the cells allowing for expansion and selection of the transduced pool. The pools were single cell cloned and subsequent clones underwent a systematic, rigorous quality control evaluation culminating with another round of exome sequencing before clones were selected to be part of the MEF 2.0 panel. Clones contained in this new panel were confirmed to be diploid, had fewer integrations than the initial panel, and performed similarly on proliferation assays as well as epidermal growth factor stimulation signaling assays. The new panel has successfully recapitulated the initial panel, but in a far more stable and reproducible cellular background. Instructions to obtain the cell line panel will be presented on the Ras Initiative website at www.cancer.gov and these cells are scheduled to become available to the Ras research community late in the spring of 2023. Citation Format: William E. Burgan, Nicole Fer, Katie Powell, Bingfang Xu, Mackenzie Meyer, Scott Eury, Abigail Neish, Daniel Soppet, Dominic Esposito, Frank McCormick, Dwight V. Nissley. An isogenic H/N/KRAS-less mouse embryonic fibroblast cell line panel derived from a size sorted diploid clonal parent [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A028.

Abstract 45: Elephant p53 protects mice from carcinogen induced death

Abstract Elephants naturally have low rates of cancer, potentially due to evolved genetic changes in tumor suppressor elephant TP53 (EP53) and amplification of 19 TP53 retrogenes. A better understanding of the mechanisms of cancer suppression in elephants by EP53 and its retrogenes could lead to more effective human cancer therapeutics. EP53 induces a strong apoptotic response compared to human TP53, especially when combined with EP53-RETROGENE 9 (EP53-R9). EP53-R9 encodes a truncated p53 protein that induces apoptosis of human cancer cells independent of EP53 through a transcription-independent mechanism. To characterize EP53 and EP53-R9’s role in cancer suppression, transgenic mice were generated to replace mouse TRP53 with EP53. Additionally, a tetracycline inducible EP53-R9 gene was inserted into a safe harbor locus in mice. Carcinogenesis studies with 3-Methylcholanthene injection revealed that EP53 mice survived significantly longer compared to heterozygous or homozygousTRP53 mice (p &amp;lt;0.0001, 0.0102). Experiments to assess the protective role of EP53-R9 alone and combined with EP53 are ongoing. Several mouse embryonic fibroblast (MEF) cell lines were generated from these transgenic mice with 14 distinct genotypes with different combinations of TRP53, EP53, and EP53-R9. p53 target gene expression studies showed that MEFs with EP53 induce higher expression of MDM2 and p21 compared to TRP53-containing MEFs, suggesting that the cancer-protective effect observed in EP53 mice is due, in part, to greater activation of p53 signaling. These results support the need for future work to assess the potential of EP53-based therapeutics for human cancer. Citation Format: Lisa M. Abegglen, Jared S. Fowles, Aidan J. Preston, Aaron Rogers, Niraja Bhachech, Brayden B. Barney, Ryan Kennington, David H. Lum, Gareth Mitchell, Joshua D. Schiffman. Elephant p53 protects mice from carcinogen induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 45.

Caffeine and Cationic Copolymers with Antimicrobial Properties.

One of the primary global health concerns is the increase in antimicrobial resistance. Polymer chemistry enables the preparation of macromolecules with hydrophobic and cationic side chains that kill bacteria by destabilizing their membranes. In the current study, macromolecules are prepared by radical copolymerization of caffeine methacrylate as the hydrophobic monomer and cationic- or zwitterionic-methacrylate monomers. The synthesized copolymers bearing tert-butyl-protected carboxybetaine as cationic side chains showed antibacterial activity toward Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). By tuning the hydrophobic content, we prepared copolymers with optimal antibacterial activity against S. aureus, including methicillin-resistant clinical isolates. Moreover, the caffeine-cationic copolymers presented good biocompatibility in a mouse embryonic fibroblast cell line, NIH 3T3, and hemocompatibility with erythrocytes even at high hydrophobic monomer content (30-50%). Therefore, incorporating caffeine and introducing tert-butyl-protected carboxybetaine as a quaternary cation in polymers could be a novel strategy to combat bacteria.

In vitro tests to evaluate embryotoxicity and irritation of Chinese herbal medicine (Pentaherbs formulation) for atopic dermatitis

Ethnopharmacological relevanceAtopic dermatitis (AD) is a common chronic inflammatory skin disorder and its prevalence is increasing in the last few decades. No treatment can cure the condition. Pregnancy often worsens the clinical manifestation. There are considerable interests in Chinese Herbal Medicine (CHM) as an alternative treatment for AD. A well tolerated CHM formula (Pentaherbs formulation, PHF) has been proven efficacious in improving life quality and reducing topical corticosteroid use in children with moderate-to-severe AD. However, safety data of PHF are not available. Aim of the studyOur study aimed to evaluate the safety of PHF and its 5 individual herbal extracts, including embryotoxicity by Embryonic Stem Cell Test (EST) and irritation by Skin Irritation Test (SIT). Materials and methodsQuality of 5 herbal extracts of PHF was confirmed by chromatography. In EST, mouse embryonic stem cell line (D3) and mouse fibroblast cell line (3T3) were used to study potential embryotoxicity. Three endpoints were assessed by concentration-response curves after 10 days’ culture: 50% inhibition of D3 differentiation into beating cardiomyocytes (ID50D3), 50% cytotoxic effects on D3 (IC50D3) and on fibroblasts (IC503T3). A biostatistically based prediction model (PM) was applied to predict the embryotoxic potentials of each CHM. In SIT, epidermis equivalent commercially available kits (EpiDerm™) were used, and concentration-viability curves were obtained by MTT assay to detect skin irritations of each CHM. ResultsChemical authentication confirmed that 5 test herbal extracts contained their main active compounds. EST results indicated that the formula PHF and its individual CHMs were non-embryotoxic, except one CHM, Amur Corktree Bark (Huang Bai, Phellodendron chinense C.K.Schneid), was weakly embryotoxic. SIT results showed that cell viability was above 50% after treatment with different concentrations of all tested CHMs. ConclusionsOur in vitro tests provided preliminary evidence for safety of the formula PHF in embryonic stem cell test and skin irritation model, but PHF shall be cautiously used in pregnant women with AD. Further studies are needed to support its clinical application as an alternative treatment for AD, especially to the patients who plan for pregnancy or at lactation stages.

Whole-cell energy modeling reveals quantitative changes of predicted energy flows in RAS mutant cancer cell lines

Cellular utilization of available energy flows to drive a multitude of forms of cellular "work" is a major biological constraint. Cells steer metabolism to address changing phenotypic states but little is known as to how bioenergetics couples to the richness of processes in a cell as a whole. Here, we outline a whole-cell energy framework that is informed by proteomic analysis and an energetics-based gene ontology. We separate analysis of metabolic supply and the capacity to generate high-energy phosphates from a representation of demand that is built on the relative abundance of ATPases and GTPases that deliver cellular work. We employed mouse embryonic fibroblast cell lines that express wild-type KRAS or oncogenic mutations and with distinct phenotypes. We observe shifts between energy-requiring processes. Calibrating against Seahorse analysis, we have created a whole-cell energy budget with apparent predictive power, for instance in relation to protein synthesis.

Metformin Improves Burn Wound Healing by Modulating Microenvironmental Fibroblasts and Macrophages.

Metformin, a biguanide, exerts different functions through various signaling pathways. In order to investigate the function and mechanism of metformin in burn wounds, we established burn rat models, subcutaneously injected metformin to treat the wounds, and observed the morphologies and the expression of collagen I, collagen III, fibronectin, and pro-inflammatory markers. In vitro experiments were performed to investigate the effects of metformin on the proliferation, migration, and collagen I synthesis of the mouse embryonic fibroblast (NIH 3T3) cell line and on the proliferation, apoptosis, and immune response of the mouse mononuclear macrophage (RAW 264.7) cell line. Finally, we studied the regulatory effects of metformin on a co-culture of RAW 264.7/NIH 3T3 cells. We found that 100 mM of metformin reduced dermal thickness, collagen I deposition, and mRNA expression of IL1β and CCL2 in rat burn wounds. In vitro experiments revealed that metformin inhibited the proliferation of NIH 3T3 and RAW 264.7 cells. Metformin attenuated NIH 3T3 cell migration via the AMPK/mTOR pathway and attenuated collagen I synthesis through the TGFβ1/Smad3 pathway. Metformin inhibited the apoptosis of RAW 264.7 cells induced by 10 μg/mL LPS. Metformin downregulated the mRNA expression of IL1β and CCL2 in RAW 264.7 cells under 1 μg/mL LPS induction by inhibiting NF-κB p65 phosphorylation. In a RAW 264.7/NIH 3T3 co-culture, metformin attenuated collagen I synthesis in NIH 3T3 cells by inhibiting RAW 264.7 paracrine secretion of TGF-β1. This provides new evidence related to the development of metformin for potentially improving burn wound healing.

Evaluation of oxindole derivatives as a potential anticancer agent against breast carcinoma cells: In vitro, in silico, and molecular docking study

In this study we have performed the in vitro anticancer activity of spiro oxindole derivatives against MCF-7 (human Adreno carcinoma) and MDA-MB-231 (triple negative breast cancer) cell lines to propose a possible role of these derivatives in the treatment of cancer. Compound 6, which has an N-benzyl substitution with a chloro group on the indolin-2-one scaffold, had the most potent activity against MCF-7 (3.55 ± 0.49 μM) and MDA-MB-231 (4.40 ± 0.468 μM) of all the synthesized molecules. A normal mouse embryonic fibroblast (NIH/3 T3) cell line was used to test the cellular toxicity of these derivatives. The results showed that none of the compounds were cytotoxic to normal cells. In addition, pharmacokinetic (ADME) and toxicity study profiles were predicted in silico. All the synthesized derivatives (1 to 7) demonstrated the necessary physicochemical properties for bioavailability. Finally, in vitro results of promising compound 6 were validated using molecular docking and dynamic simulation studies, which revealed their binding affinities and conformational stability in the binding cavity. Thus, these derivatives may serve as lead structures for a new generation of anticancer agents.