Mouse Embryonic Fibroblasts Research Articles

Design, Synthesis, and Evaluation of a New Series of Hydrazones as Small-Molecule Akt Inhibitors for NSCLC Therapy.

In an endeavor to identify small molecules for the management of non-small-cell lung carcinoma, 10 new hydrazone derivatives (3a-j) were synthesized. MTT test was conducted to examine their cytotoxic activities against human lung adenocarcinoma (A549) and mouse embryonic fibroblast (L929) cells. Compounds 3a, 3e, 3g, and 3i were determined as selective antitumor agents on A549 cell line. Further studies were conducted to figure out their mode of action. Compounds 3a and 3g markedly induced apoptosis in A549 cells. However, both compounds did not show any significant inhibitory effect on Akt. On the other hand, in vitro experiments suggest that compounds 3e and 3i are potential anti-NSCLC agents acting through Akt inhibition. Furthermore, molecular docking studies revealed a unique binding mode for compound 3i (the strongest Akt inhibitor in this series), which interacts with both hinge region and acidic pocket of Akt2. However, it is understood that compounds 3a and 3g exert their cytotoxic and apoptotic effects on A549 cells via different pathway(s).

LRP1 Deficiency Promotes Mitostasis in Response to Oxidative Stress: Implications for Mitochondrial Targeting after Traumatic Brain Injury.

The brain undergoes oxidative stress and mitochondrial dysfunction following physiological insults such as Traumatic brain injury (TBI), ischemia-reperfusion, and stroke. Pharmacotherapeutics targeting mitochondria (mitoceuticals) against oxidative stress include antioxidants, mild uncouplers, and enhancers of mitochondrial biogenesis, which have been shown to improve pathophysiological outcomes after TBI. However, to date, there is no effective treatment for TBI. Studies have suggested that the deletion of LDL receptor-related protein 1 (LRP1) in adult neurons or glial cells could be beneficial and promote neuronal health. In this study, we used WT and LRP1 knockout (LKO) mouse embryonic fibroblast cells to examine mitochondrial outcomes following exogenous oxidative stress. Furthermore, we developed a novel technique to measure mitochondrial morphometric dynamics using transgenic mitochondrial reporter mice mtD2g (mitochondrial-specific Dendra2 green) in a TBI model. We found that oxidative stress increased the quantity of fragmented and spherical-shaped mitochondria in the injury core of the ipsilateral cortex following TBI, whereas rod-like elongated mitochondria were seen in the corresponding contralateral cortex. Critically, LRP1 deficiency significantly decreased mitochondrial fragmentation, preserving mitochondrial function and cell growth following exogenous oxidative stress. Collectively, our results show that targeting LRP1 to improve mitochondrial function is a potential pharmacotherapeutic strategy against oxidative damage in TBI and other neurodegenerative diseases.

Generation of CRISPR-Cas9-mediated knockin mutant models in mice and MEFs for studies of polymorphism in clock genes

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in mouse embryonic fibroblasts (MEFs) as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Quantification of the mutation frequency in bulk MEF populations provides a valuable basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.

Alleviation of neurotoxicity induced by polystyrene nanoplastics by increased exocytosis from neurons

Nanoplastics (NPs) are potentially toxic and pose a health risk as they can induce an inflammatory response and oxidative stress at cellular and organismal levels. Humans can be exposed to NPs through various routes, including ingestion, inhalation, and skin contact. Notably, uptake into the body via inhalation could result in brain accumulation, which may occur directly across the blood-brain barrier or via other routes. NPs that accumulate in the brain may be endocytosed into neurons, inducing neurotoxicity. Recently, we demonstrated that exposure to polystyrene (PS)-NPs reduces the viability of neurons. We have also reported that inhibiting the retrograde transport of PS-NPs by histone deacetylase 6 (HDAC6) prevents their intracellular accumulation and promotes their export in mouse embryonic fibroblasts. However, whether HDAC6 inhibition can improve neuronal viability by increasing exocytosis of PS-NPs from neurons remains unknown. In this study, mice were intranasally administered fluorescent PS-NPs (PS-YG), which accumulated in the brain and showed potential neurotoxic effects. In cultured neurons, the HDAC6 inhibitor ACY-1215 reduced the fluorescence signal detected from PS-YG, suggesting that the removal of PS-YG from neurons was promoted. Therefore, these results suggest that blocking the retrograde transport of PS-NPs using an HDAC6 inhibitor can alleviate the neurotoxic effects of PS-NPs that enter the brain.

Tannic acid-loaded chitosan-RGD-alginate scaffolds for wound healing and skin regeneration

Hydrogels have drawn much attention in the field of tissue regeneration and wound healing owing to the application of biocompatible peptides to tailor structural features necessitating optimal tissue remodeling performance. In the current study, polymers and peptide were explored to develop scaffolds for wound healing and skin tissue regeneration. Alginate (Alg), chitosan (CS), and arginine-glycine-aspartate (RGD) were used to fabricate composite scaffolds crosslinked with tannic acid (TA), which also served as a bioactive. The use of RGD transformed the physicochemical and morphological features of the 3D scaffolds and TA crosslinking of the scaffolds improved their mechanical properties, specifically tensile strength, compressive Young’s modulus, yield strength, and ultimate compressive strength. The incorporation of TA as both a crosslinker and a bioactive allowed for 86% encapsulation efficiency and burst release of 57% of TA in 24 h, accompanied by an 8.5% steady release per day of up to 90% over 5 d. The scaffolds increased mouse embryonic fibroblast cell viability over 3 d, progressing from slightly cytotoxic to non-cytotoxic (cell viability >90%). Wound closure and tissue regeneration evaluations in a SpragueDawley rat wound model at predetermined wound healing time points highlighted the superiority of the Alg-RGD-CS and Alg-RGD-CS-TA scaffolds over the commercial comparator product and control. The scaffolds’ superior performance included accelerated tissue remodeling performance from the early to the late stages of wound healing, indicated by the lack of defects and scarring in scaffold-treated tissues. This promising performance supports the design of wound dressings that can act as delivery systems for the treatment of acute and chronic wounds.

Control of enhancer and promoter activation in the type I interferon response by the histone demethylase Kdm4d/JMJD2d.

Transcriptional activation depends on the interplay of chromatin modifiers to establish a permissive epigenetic landscape. While histone 3 lysine 9 (H3K9) methylation has long been associated with gene repression, there is limited evidence to support a role for H3K9 demethylases in gene activation. We leveraged knockdown and overexpression of JMJD2d / Kdm4d in mouse embryonic fibroblasts, coupled with extensive epigenomic analysesm to decipher the role of histone 3 lysine 9 demethylases in the innate immune response. Here we describe the H3K9 demethylase Kdm4d/JMJD2d as a positive regulator of type I interferon responses. In mouse embryonic fibroblasts (MEFs), depletion of JMJD2d attenuates the transcriptional response, conferring increased viral susceptibility, while overexpression of the demethylase results in more robust IFN activation. We find that the underlying mechanism of JMJD2d in type I interferon responses consists of an effect both on the transcription of enhancer RNAs (eRNAs) and on dynamic H3K9me2 at associated promoters. In support of these findings, we establish that JMJD2d is associated with enhancer regions throughout the genome prior to stimulation but is redistributed to inducible promoters in conjunction with transcriptional activation. Taken together, our data reveal JMJD2d as a chromatin modifier that connects enhancer transcription with promoter demethylation to modulate transcriptional responses.

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.

Engineering preparation and sustained delivery of bone functional exosomes-laden biodegradable hydrogel for in situ bone regeneration

As an emerging cell-free therapy, exosomes derived from stem cells have been widely explored for tissue regeneration. However, it is still a great challenge to achieve the engineering extraction of bone-functionalized exosomes and their sustained delivery in tissue defects. Herein, a novel bone morphogenetic protein 2 (BMP2)-overexpressed engineered exosomes-loaded GelMA hydrogel was rationally constructed for in-situ bone regeneration. BMP2-overexpressed exosomes were engineering prepared using mouse embryonic fibroblasts (NIH-3T3) as tool cells via gene manipulations and ultracentrifugation techniques. The sustained delivery of exosomes in cranial defects was achieved by loading GelMA biodegradable hydrogel. In addition, the pre-prepared BMP2-overexpressed exosome-loaded GelMA hydrogel showed excellent porous structure, rheological property and biocompatibility. In vitro biological results indicated that BMP2-overexpressed exosomes significantly promoted proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs). In vivo animal experiments analysis revealed BMP2-overexpressed exsomes-laden biodegradable hydrogel effectively improved in situ bone regeneration via mice cranial defect model. Overall, this study provided a promising cell-free therapy of engineered exosomes-loaded biodegradable hydrogel for treating bone defects.

Osteopontin Is an Integral Mediator of Cardiac Interstitial Fibrosis in Models of Human Immunodeficiency Virus Infection.

People with human immunodeficiency virus (HIV) have heightened incidence/risk of diastolic dysfunction and heart failure. Women with HIV have elevated cardiac fibrosis, and plasma osteopontin (Opn) is correlated to cardiac pathology. Therefore, this study provides mechanistic insight into the relationship between osteopontin and cardiac fibrosis during HIV infection. Mouse embryonic fibroblasts (MEFs) modeled cardiac fibroblasts in vitro. Simian immunodeficiency virus (SIV)-infected macaques with or without antiretroviral therapy and HIV-infected humanized mice modeled HIV-associated cardiac fibrosis. Lipopolysaccharide-stimulated MEFs were myofibroblast-like, secreted cytokines, and produced Opn transcripts. SIV-infected animals had elevated plasma Opn at necropsy, full-length Opn in the ventricle, and ventricular interstitial fibrosis. Regression modeling identified growth differentiation factor 15, CD14+CD16+ monocytes, and CD163 expression on CD14+CD16+ monocytes as independent predictors of plasma Opn during SIV infection. HIV-infected humanized mice showed increased interstitial fibrosis compared to uninfected/untreated animals, and systemic inhibition of osteopontin by RNA aptamer reduced left ventricle fibrosis in HIV-infected humanized mice. Since Opn is elevated in the plasma and left ventricle during SIV infection and systemic inhibition of Opn reduced cardiac fibrosis in HIV-infected mice, Opn may be a potential target for adjunctive therapies to reduce cardiac fibrosis in people with HIV.

Construction of a bioactive copper-based metal organic framework-embedded dual-crosslinked alginate hydrogel for antimicrobial applications

Metal-organic frameworks (MOFs) containing bioactive metals have the potential to exhibit antimicrobial activity by releasing metal ions or ligands through the cleavage of metal-ligand bonds. Recently, copper-based MOFs (Cu-MOFs) with sustained release capability, porosity, and structural flexibility have shown promising antimicrobial properties. However, for clinical use, the controlled release of Cu2+ over an extended time period is crucial to prevent toxicity. In this study, we developed an alginate-based antimicrobial scaffold and encapsulated MOFs within a dual-crosslinked alginate polymer network. We synthesized Cu-MOFs containing glutarate (Glu) and 4,4′-azopyridine (AZPY) (Cu(AZPY)-MOF) and encapsulated them in an alginate-based hydrogel through a combination of visible light-induced photo and calcium ion-induced chemical crosslinking processes. We confirmed Cu(AZPY)-MOF synthesis using scanning electron microscopy, transmission electron microscopy, powder X-ray diffraction, and thermogravimetric analysis. This antimicrobial hydrogel demonstrated excellent antibacterial and antifungal properties against two bacterial strains (MRSA and S. mutans, with >99.9 % antibacterial rate) and one fungal strain (C. albicans, with >78.7 % antifungal rate) as well as negligible cytotoxicity towards mouse embryonic fibroblasts, making it a promising candidate for various tissue engineering applications in biomedical fields.

A Series of Non-Oxido VIV Complexes of Dibasic ONS Donor Ligands: Solution Stability, Chemical Transformations, Protein Interactions, and Antiproliferative Activity.

A series of mononuclear non-oxido vanadium(IV) complexes, [VIV(L1-4)2] (1-4), featuring tridentate bi-negative ONS chelating S-alkyl/aryl-substituted dithiocarbazate ligands H2L1-4, are reported. All the synthesized non-oxido VIV compounds are characterized by elemental analysis, spectroscopy (IR, UV-vis, and EPR), ESI-MS, as well as electrochemical techniques (cyclic voltammetry). Single-crystal X-ray diffraction studies of 1-3 reveal that the mononuclear non-oxido VIV complexes show distorted octahedral (1 and 2) or trigonal prismatic (3) arrangement around the non-oxido VIV center. EPR and DFT data indicate the coexistence of mer and fac isomers in solution, and ESI-MS results suggest a partial oxidation of [VIV(L1-4)2] to [VV(L1-4)2]+ and [VVO2(L1-4)]-; therefore, all these three complexes are plausible active species. Complexes 1-4 interact with bovine serum albumin (BSA) with a moderate binding affinity, and docking calculations reveal non-covalent interactions with different regions of BSA, particularly with Tyr, Lys, Arg, and Thr residues. In vitro cytotoxic activity of all complexes is assayed against the HT-29 (colon cancer) and HeLa (cervical cancer) cells and compared with the NIH-3T3 (mouse embryonic fibroblast) normal cell line by MTT assay and DAPI staining. The results suggest that complexes 1-4 are cytotoxic in nature and induce cell death in the cancer cell lines by apoptosis and that a mixture of VIV, VV, and VVO2 species could be responsible for the biological activity.

Synthesis and Antitumor Evaluation of Biotin-SN38-Valproic Acid Conjugates.

Despite the strong anticancer activity of SN38 (7-ethyl-10-hydroxy-camptothecin), the severe side effects and loss of anticancer activity caused by the lack of selectivity to cancer cells and hydrolysis of ring E prevent its clinical application. To address the issue, herein a multifunctional SN38 derivative (compound 9) containing biotin (tumor-targeting group) and valproic acid (histone deacetylase inhibitor, HDACi) was synthesized via click chemistry and evaluated using MTT assay. The in vitro cytotoxicity study showed that compound 9 exhibited superior cytotoxicity than irinotecan against human cervical cancer HeLa cells, albeit it was inferior to SN38. More significantly, compound 9 significantly reduced toxicity in mouse embryonic fibroblast NIH3T3 cells, indicating that compound 9 had the capacity to enhance tumor targeting due to its cell selectivity. Further studies demonstrated that, compared with irinotecan, compound 9 induced similar apoptosis of cancer cells. Consequently, compound 9 can not only improve its tumor-targeting ability mediated by biotin but also exert potent anticancer activity through the effect of SN38 and valproic acid, indicating that the design concept is an effective strategy for the structural modification of SN38.

In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.

In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

MTFP1 loss sensitizes to mtDNA-driven sterile inflammation and heart failure in mice

Mitochondrial integrity is paramount to heart homeostasis. Under certain stress conditions, mitochondrial DNA (mtDNA) can leak into the cytosol and trigger type I innate immunity via the DNA sensing cGAS-STING pathway, the downstream interferon (IFN) regulatory factor 3 (IRF3) and cell surface receptor IFNAR1. Our previous work shows that loss of the inner membrane (IM) protein Mitochondrial Fission Process 1 (MTFP1) in cardiomyocytes of mice (cMKO) disturbs mitochondrial IM integrity leading to the activation of immune responses and a progressive dilated cardiomyopathy (DCM), which culminates into heart failure (HF) and middle-aged death. Investigate the impact of MTFP1 loss on mtDNA driven type I immune response and inflammation. In vitro, Mtfp1-/- mouse embryonic fibroblasts (MEFs) depleted or not of mtDNA, Sting or Irf3 were subjected to mitochondrial outer membrane permeabilization to assess the Interferons Stimulated Genes (ISGs) expression and IRF3 phosphorylation. In vivo, cMKO mice were additionally deleted of Sting or Ifnar1 and subjected to echocardiography to assess DCM progression. Upon treatment, Mtfp1-/- MEFs display increased ISGs expression and IRF3 phosphorylation compared to WT cells, which is abrogated by the depletion of mtDNA, STING or IRF3 in these cells. In vivo, whole body deletion of STING in cMKO mice mitigates HF, whereas IFNAR1 ablation fully rescues cardiac dysfunction and remodeling and extends lifespan of mice. Collectively, our data suggest a role of MTFP1 in restraining mtDNA-dependent inflammation, which in vivo fosters HF in the cMKO mice.

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.

Imaging Subcellular AMPK Activity Using an Excitation‐Ratiometric AMPK Activity Reporter

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a master regulator of cellular metabolism, phosphorylating a variety of downstream targets throughout the cell. Subcellular AMPK activity results in regulation of glycolysis, lipid and protein biosynthesis, mitochondrial function, and gene expression. But how AMPK senses and responds to stimuli in a compartment-specific manner is not well understood, leaving an incomplete picture of compartmentalized AMPK activity. Key tools for studying subcellular AMPK activity are genetically encoded AMPK activity reporters (AMPKARs), which allow for the quantitative visualization of subcelluar AMPK activity. However, many AMPKARs suffer from poor dynamic range and sensitivity, limiting their application. I recently reported the development of a new excitation-ratiometric (ExRai) AMPKAR, a single-fluorophore AMPKAR with enhanced dynamic range for detection of subtle, subcellular AMPK activity. I used ExRai AMPKAR to study subcellular AMPK activity at several locations, including the lysosome and mitochondria, identifying new mechanisms for the regulation of AMPK activity. Here, I describe the use of ExRai AMPKAR to image subcellular AMPK activity in mouse embryonic fibroblasts using both widefield and confocal microscopy. I also describe the culture of mouse embryonic fibroblasts. Through the use of ExRai AMPKAR, subcellular AMPK activity can be illuminated to better understand how this central kinase regulates cellular metabolism. © 2023 The Author. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Imaging subcellular AMPK activity using ExRai AMPKAR Support Protocol 1: Culturing of mouse embryonic fibroblasts for live-cell imaging Support Protocol 2: Live-cell imaging of ExRai AMPKAR using confocal microscopy.

Red clover (Trifolium pratense L.) extract inhibits ferroptotic cell death by modulating cellular iron homeostasis.

Red clover (Trifolium pratense L.) is a traditional Chinese medicine and use as herbal medicine which has the effects of regulating menopausal symptoms, heart problem, inflammatory disease, psoriasis and cognitive deficits. In previous reported, the studies of red clover were mainly focused on clinical practice. the pharmacological functions of red clover not fully elucidated. To identify the molecules that regulate ferroptosis, we examined whether red clover (Trifolium pratense L.) extracts (RCE) affected ferroptosis induced by chemical treatment or cystine/glutamate antiporter (xCT) deficiency. Cellular models for ferroptosis were induced by erastin/Ras-selectiv lethal 3 (RSL3) treatment or xCT deficiency in mouse embryonic fibroblasts (MEFs). Intracellular iron and peroxidized lipid levels were determined using Calcein-AM and BODIPY-C11 fluorescence dyes, respectively. Protein and mRNA were quantified by Western blot and real-time polymerase chain reaction, respectively. RNA sequencing analysis was performed on xCT-/- MEFs. RCE significantly suppressed ferroptosis induced by both erastin/RSL3 treatment and xCT deficiency. The anti-ferroptotic effects of RCE correlated to ferroptotic phenotypic changes such as cellular iron accumulation and lipid peroxidation in cellular ferroptosis models. Importantly, RCE affected levels of iron metabolism-related proteins including iron regulatory protein 1, ferroportin 1 (FPN1), divalent metal transporter 1, and transferrin receptor. RNA sequencing analysis of xCT-/- MEFs identified that expression of cellular defense genes was upregulated, while expression of cell death-related genes was downregulated, by RCE. RCE potently suppressed ferroptosis triggered both by erastin/RSL3 treatment and xCT deficiency by modulating cellular iron homeostasis. This is the first report that RCE has therapeutic potential in diseases associated with ferroptotic cell death, particularly ferroptosis induced by dysregulation of cellular iron metabolism.

Pivotal role for the non-receptor tyrosine kinase SRMS in IRF3-dependent innate antiviral immune response in murine cells

Abstract Upon viral infections, RIG-I-like receptors (RLRs) sense cytosolic viral RNAs and initiate signaling pathways culminating in the activation of IRF3 and subsequent type I interferon (IFN) antiviral response. This intrinsic protective mechanism is orchestrated by a host of positive and negative regulators, which collectively govern the magnitude and duration of antiviral signaling. SRMS is an understudied non-receptor tyrosine kinase frequently overexpressed in breast cancers. SRMS restrains autophagy and promotes tumor growth, although its physiological function is unclear. We report here that SRMS is critical for IRF3-dependent innate antiviral response in murine cells. Depletion of SRMS impaired virus- or dsRNA-induced expression of type I and III IFNs and ISGs in mouse embryonic fibroblasts (MEFs), mouse hepatoma hepa1–6 cells, and primary mouse lung fibroblasts, concomitant with heightened viral replication. Mechanistically, SRMS loss led to diminished phosphorylations of TBK1 and IRF3, IRF3 dimerization and nuclear translocation. The kinase activity of SRMS, however, was dispensable. Reconstituting the expression of SRMS of mouse or human origin, be it wild-type or kinase-dead protein, in SRMS-null MEFs restored antiviral signaling. Autophagy regulation was not involved, either. Interestingly, antiviral gene expression via the RLRs in human U2OS and MDA-MB-231 cells was not impacted by SRMS deletion, suggesting signaling redundancy in human cells. Altogether, our data uncover a novel role of SRMS in controlling IRF3-dependent innate antiviral responses and describe a non-canonical, kinase- and autophagy-independent regulatory mechanism that operates differentially between mouse and human species. R21 AI142044

CRISPR/Cas9 Induced Duplications, Deletions and Inversions in Mouse Zygotes Lead to Karyotype Instability

CRISPR/Cas9 technology has been widely used for targeted modification of the mammalian genomes. We have analyzed the karyotype of 18 mouse fibroblast cell lines with Cntn6 gene rearrangements introduced by CRISPR/Cas9. We have produced cell lines with 2374 kb Cntn6 gene duplications, 1137 kb deletions and inversions of similar size. In addition, we have performed cytogenetic analysis for five control mouse embryonic fibroblasts with the intact Cntn6 gene alleles. The cell lines heterozygous for Cntn6 gene inversion and homozygous and heterozygous for Cntn6 gene duplication had a high level of polyploidy (20–46%), as well as chromosome 6 monosomy (1–9%) and trisomy (1–8%). No trisomy was detected in the four cell lines with the deletion and duplication of the Cntn6 gene in the compound, and the proportion of polyploid cells was minimal (1.5–5.7%). Thus, we have shown the karyotype destabilization in the cell lines that have undergone genome editing using CRISPR/Cas9 system.

UV-Accelerated Synthesis of Gold Nanoparticle-Pluronic Nanocomposites for X-ray Computed Tomography Contrast Enhancement.

Eco-friendly chemical methods using FDA-approved Pluronic F127 (PLU) block copolymer have garnered much attention for simultaneously forming and stabilizing Au nanoparticles (AuNPs). Given the remarkable properties of AuNPs for usage in various fields, especially in biomedicine, we performed a systematic study to synthesize AuNP-PLU nanocomposites under optimized conditions using UV irradiation for accelerating the reaction. The use of UV irradiation at 254 nm resulted in several advantages over the control method conducted under ambient light (control). The AuNP-PLU-UV nanocomposite was produced six times faster, lasting 10 min, and exhibited lower size dispersion than the control. A set of experimental techniques was applied to determine the structure and morphology of the produced nanocomposites as affected by the UV irradiation. The MTT assay was conducted to estimate IC50 values of AuNP-PLU-UV in NIH 3T3 mouse embryonic fibroblasts, and the results suggest that the sample is more compatible with cells than control samples. Afterward, in vivo maternal and fetal toxicity assays were performed in rats to evaluate the effect of AuNP-PLU-UV formulation during pregnancy. Under the tested conditions, the treatment was found to be safe for the mother and fetus. As a proof of concept or application, the synthesized Au:PLU were tested as contrast agents with an X-ray computed tomography scan (X-ray CT).