Eukaryotic Translation Initiation Factor 2α Research Articles

GADD34 suppresses eIF2α phosphorylation and improves cognitive function in Alzheimer's disease-model mice

Alzheimer's disease (AD) causes neurodegeneration, leading to cognitive impairment and memory loss. Our previous studies have demonstrated that the induction of growth arrest and DNA damage-inducible gene 34 (GADD34) by quercetin can affect eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-activated transcription factor 4 (ATF4) signaling. However, the relationship between GADD34 expression and cognitive function has not been clarified. In this study, we determined the direct effect of GADD34 on memory. To achieve this, truncated GADD34 (GADD34.5) was injected into the mouse brain to suppress eIF2α phosphorylation and evaluate memory. The injection of GADD34.5 into the hippocampus in AD-model mice did not improve novel object recognition but improved novel object location. The injection of GADD34.5 into the amygdala also resulted in the maintenance of contextual fear memory based on the fear condition test. These results suggest that GADD34 is effective in improving memory for spatial cognition and contextual fear conditioning in AD by inhibiting eIF2α phosphorylation. In summary, GADD34 suppresses eIF2α phosphorylation in the brain and prevents memory loss. As quercetin feeding increases GADD34 expression, it might be used in preventative applications for AD.

Self-immolative polymer-based immunogenic cell death inducer for regulation of redox homeostasis

Doxorubicin (DOX), widely used as an anticancer drug, is considered an immunogenic cell death (ICD) inducer that enhances cancer immunotherapy. However, its extended application as an ICD inducer has been limited owing to poor antigenicity and inefficient adjuvanticity. To enhance the immunogenicity of DOX, we prepare a reactive oxygen species (ROS)-responsive self-immolative polymer (R–SIP) that can efficiently destroy redox homeostasis via self-immolation-mediated glutathione depletion in cancer cells. Owing to its amphiphilic nature, R–SIP self-assemble into nano-sized particles under aqueous conditions, and DOX is efficiently encapsulated inside the nanoparticles by a simple dialysis method. Interestingly, when treated with 4T1 cancer cells, DOX-encapsulated R–SIP (DR-SIP) induces the phosphorylation of eukaryotic translation initiation factor 2α and overexpression of ecto-calreticulin, resulting in endoplasmic reticulum-associated ICD. In addition, DR-SIP contributes to the maturation of dendritic cells by promoting the release of damage-associated molecular patterns (DAMPs) from cancer cells. When intravenously administered to tumor-bearing mice, DR-SIP remarkably inhibits tumor growth compared with DOX alone. Overall, DR-SIP may have the potential to elicit an immune response as an ICD inducer.

Exosome-transmitted circCABIN1 promotes temozolomide resistance in glioblastoma via sustaining ErbB downstream signaling

Although temozolomide (TMZ) provides significant clinical benefit for glioblastoma (GBM), responses are limited by the emergence of acquired resistance. Here, we demonstrate that exosomal circCABIN1 secreted from TMZ-resistant cells was packaged into exosomes and then disseminated TMZ resistance of receipt cells. CircCABIN1 could be cyclized by eukaryotic translation initiation factor 4A3 (EIF4A3) and is highly expressed in GBM tissues and glioma stem cells (GSCs). CircCABIN1 is required for the self-renewal maintenance of GSCs to initiate acquired resistance. Mechanistically, circCABIN1 regulated the expression of olfactomedin-like 3 (OLFML3) by sponging miR-637. Moreover, upregulation of OLFML3 activating the ErbB signaling pathway and ultimately contributing to stemness reprogramming and TMZ resistance. Treatment of GBM orthotopic mice xenografts with engineered exosomes targeting circCABIN1 and OLFML3 provided prominent targetability and had significantly improved antitumor activity of TMZ. In summary, our work proposed a novel mechanism for drug resistance transmission in GBM and provided evidence that engineered exosomes are a promising clinical tool for cancer prevention and therapy.Graphical

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A

AbstractMetabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1–dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

Ciclopirox Inhibition of eIF5A Hypusination Attenuates Fibroblast Activation and Cardiac Fibrosis.

Cardiac fibrosis is a primary contributor to heart failure (HF), and is considered to be a targetable process for HF therapy. Cardiac fibroblast (CF) activation accompanied by excessive extracellular matrix (ECM) production is central to the initiation and maintenance of fibrotic scarring in cardiac fibrosis. However, therapeutic compounds targeting CF activation remain limited in treating cardiac fibrosis. Eukaryotic translation initiation factor 5A (eIF5A), upon being hypusinated, is essential for the translation elongation of proline-codon rich mRNAs. In this study, we found that increased hypusinated eIF5A protein levels were associated with cardiac fibrosis and heart dysfunction in myocardial infarction (MI) mouse models. Ciclopirox (CPX), an FDA-approved antifungal drug, inhibits the deoxyhypusine hydroxylase (DOHH) enzyme required for eIF5A hypusination. Results from preventive and reversal mouse models suggest that CPX treatment significantly reduced MI-driven cardiac fibrosis and improved cardiac function. In vitro studies of isolated mouse primary CFs revealed that inhibition of eIF5A hypusination using CPX significantly abolished TGFβ induced CF proliferation, activation, and collagen expression. Proteomic analysis from mouse CFs reveals that CPX downregulates the expression of proline-rich proteins that are enriched in extracellular matrix and cell adhesion pathways. Our findings are relevant to human heart disease, as increased hypusinated eIF5A levels were observed in heart samples of ischemic heart failure patients compared to healthy subjects. Together, these results suggest that CPX can be repurposed to treat cardiac fibrosis and ischemic heart failure.

Identification of reference genes for RT-qPCR analysis across kiwifruit species under iron deficiency conditions

Selecting appropriate reference genes is crucial for the accuracy of gene expression analysis. Although rootstock species play vital roles in kiwifruit production, the reference genes has not been identified in kiwifruit roots and across kiwifruit species. Additionally, iron (Fe) deficiency has been frequently observed in kiwifruit orchards, but the reference genes is not validated. Here, we assessed ten candidate reference genes: actin (ACT), actin-depolymerizing factor family protein (ADF), ADP-ribosylation factor (ARF), eukaryotic translation initiation factor 5A (eIF5A), ribosomal protein L19 (RPL19), 40S ribosomal protein S17 (RPS17), suppressor of G2 allele of SKP1 (SGT1), translationally-controlled tumor protein (TCTP), translation initiation factor 2-γ (TIF2-γ) and ubiquitin-conjugating enzyme E2 variant (UBE2V), for their suitability as reliable internal controls in roots, stems and leaves of four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) under direct and bicarbonate-induced Fe deficiency conditions. The expression stability of the candidate genes was evaluated by four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Based on the comprehensive ranking, we recommended TCTP + UBE2V as the best reference genes for roots, UBE2V + ACT for stems, RPL19 + ARF for leaves, and UBE2V + RPL19 for all the tissues. The reliability of the recommended reference genes was verified by six target genes involved in Fe uptake and transport. These results provide a basis for improving the accuracy of gene expression analysis and elucidating the molecular mechanisms of Fe deficiency adaptation in kiwifruit species.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, Tcell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific Tcells was inhibited by Sephin1. A special TCR+ macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14.

This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

EIF5A2 Promotes Doxorubicin Resistance in Bladder Cancer Cells through the TGF-β Signaling Pathway.

Doxorubicin (DOX) is a commonly used chemotherapeutic agent, but bladder cancer (BC) patients often develop resistance that limits therapeutic efficacy. Recent research has demonstrated a link between medication resistance and the expression of eukaryotic translation initiation factor 5A2 (EIF5A2) in tumors. This study aimed to investigate whether EIF5A2 affects the resistance of BC cells to doxorubicin through the transforming growth factor (TGF)-β signaling pathway. Doxorubicin-resistant cells in BC (T24/DOX and 5637/DOX) were constructed, then cell viability was detected by cell counting kit-8 (CCK-8); EIF5A2 mRNA expression was detected using quantitative real-time PCR (qRT-PCR); cell proliferation was detected using clone formation; apoptosis was detected by flow cytometry; and finally, proteins related to the TGF-β signaling pathway (EIF5A2, TGF-β1, p-small mothers against decapentaplegic 2 (Smad2)/Smad2, p-Smad3/Smad3) were detected using western blot. EIF5A2 was up-regulated in DOX-resistant BC cells, and DOX intervention promoted proliferation and inhibited apoptosis in DOX-resistant BC cells. si-EIF5A2 reversed the above effects. EIF5A2 resulted in DOX resistance by activating the TGF-β pathway, and the TGF-β activator SRI-011381 reversed the inhibitory effect of si-EIF5A2 on DOX resistance. EIF5A2 promotes DOX resistance in BC cells through the TGF-β signaling pathway, and EIF5A2 may be a potential counter-resistance therapeutic strategy in BC chemotherapy.

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush.

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury. Valproate is a histone deacetylase inhibitor and multitarget drug, which has been demonstrated to protect retinal neurons. In this study, we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling. We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope. Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein, phosphorylated eukaryotic translation initiation factor 2α, and caspase-12 in the endoplasmic reticulum of retinal ganglion cells. These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress. These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

Proteomics analysis reveals the mechanism of growth retardation under a specific nitrogen environment for Caulerpa lentillifera

Caulerpa lentillifera is a macroalgae with high economic value in tropical and subtropical regions. To date, the influence of nitrogen sources on the growth of C. lentillifera is unclear, and the biological relevance behind the nonlinear growth phenomenon of C. lentillifera under different nitrogen concentrations remains unknown. In this study, besides traditional growth observation and photosynthetic pigment content analysis, a quantitative proteomics method was applied to investigate the molecular mechanisms of C. lentillifera in response to 55, 110, 220, 440, and 880 μmol/L nitrate concentrations. Our results showed that neither the number of branches nor the percentage weight gain of C. lentillifera increased linearly with nitrate concentration during the 12-day culture. Growth retardation was observed at 220 μmol/L nitrate concentration (4× condition), but the concentrations of chlorophyll a, chlorophyll b, and β-carotene extracted from C. lentillifera showed no difference under all nitrate conditions. Proteomics analysis results coupled with principal component analysis demonstrated that 4× condition is a unique culture condition for C. lentillifera. In addition, proteins with substantial expression changes were involved in different biological functions, such as protein synthesis, genetic information processing, protein ubiquitination, and cytoskeleton formation. Quantitative proteomics analysis further confirmed that 17 proteins were significantly upregulated under the 4× condition, while three proteins were downregulated, indicating that this culture condition is an environmental stress to C. lentillifera and causes growth retardation. Moreover, according to the protein–protein interaction network, eukaryotic translation initiation factor 4A-1 (eIF4A), one of 17 protein upregulated was suggested as a potential marker of the growth retardation phenomenon of C. lentillifera. Our findings provide the first molecular evidence for understanding the mechanism under different nitrate conditions and propose a potential growth marker for the cultivation of C. lentillifera.

XPO1-Mediated EIF1AX Cytoplasmic Relocation Promotes Tumor Migration and Invasion in Endometrial Carcinoma.

Dysregulation of eukaryotic translation initiation factor 1A, X-linked (EIF1AX), has been implicated in the pathogenesis of some cancers. However, the role of EIF1AX in endometrial carcinoma (EC) remains unknown. We investigated the EIF1AX expression in EC patients and assessed its tumorigenesis-associated function and nucleocytoplasmic transport mechanism in vitro and in vivo. The results indicated that the cytoplasmic EIF1AX expression showed a gradual increase when going from endometrium normal tissue, simple endometrial hyperplasia, complex endometrial hyperplasia, and endometrial atypical hyperplasia to EC, while vice versa for the nuclear EIF1AX expression. In addition, the cytoplasmic EIF1AX expression was positively correlated with histologic type, high International Federation of Gynecology and Obstetrics (FIGO) grade, advanced FIGO stage, deeper infiltration, high Ki67 index, and shorter recurrence-free survival in EC patients. In vitro, short hairpin RNA-mediated EIF1AX depletion or SV40NLS-mediated EIF1AX import into the nucleus in multiple human EC cells potently suppressed cell migration and invasion, epithelial-mesenchymal transition, and lung metastasis. Moreover, exportin 1 induced the transport of EIF1AX from the nucleus to the cytoplasm that could be inhibited by leptomycin B treatment or the mutation in the EIF1AX location sequence. These results demonstrate that cytoplasmic EIF1AX may play a key role in the incidence and promotion of EC, and thus, targeting EIF1AX or its nucleocytoplasmic transport process may offer an effective new therapeutic approach to EC.

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation.

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.

Selection and Validation of Suitable Reference Genes for Gene Expression Studies in Callerya speciosa (Champ. ex Benth.) Schot under Different Experimental Conditions

The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) strongly depends on the stability of reference gene. It is essential to select a suitable reference gene in order to obtain reliable RT-qPCR results when gene expression profiles were evaluated. Callerya speciosa, a traditional Chinese medicine, has a long cultivation history in south China. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L34), ACTIN (actin), TUA2 (tubulin alpha chain), TUB1 (tubulin beta-1 chain), TIF5 (eukaryotic translation initiation factor 5A), UBQ (polyubiquitin), and EF2 (elongation factor 2), were selected from the transcriptome databases, and their expression stability under six different experimental conditions (developmental stages, tissues, MeJA treatment, GA3 treatment, CPPU treatment, and PP333 treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, and RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the best stability under most of the hormone treatments. TUA2 and EF2 were the two least suitable ones as reference genes in C. speciosa. The expression pattern of CsMYB36, a gene associated with the regulation of isoflavonoid biosynthesis in C. speciosa, verified the accuracy of our experimental results. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa and other Callerya species in the future.

Role of Fragile X Mental Retardation protein in Amyloid Beta 1‐42 Induced Translational Suppression

AbstractBackgroundAlzheimer’s Disease (AD) is the most common cause of dementia where the accumulation of the peptide Amyloid‐Beta (Aβ) is the main theory linked to the pathogenesis of the disease. Fragile X Mental Retardation Protein (FMRP) is an RNA‐binding protein that functions to suppress global translation. Because Aβ is known to be a toxic peptide capable of inducing the Endoplasmic Reticulum (ER) stress response and because augmented translational suppression is one of the main molecular phenotypes in ER stress response, we ask whether and how FMRP is involved in Aß induced translational suppression.MethodPrimary mouse cortical neuronal cultures from wild type (WT) or Fmr1 KO pups were treated with Aβ (Aβ1–42) during 24 hours. For protein expression identification and quantification we used western blot and ImageJ software respectively. Protein translation was assessed by inhibition with puromycin. Neuronal networking assessment was made with multielectrode array system (MEA).ResultIn this study, we observed that treatments of synthetic Aβ peptides in culture primary mouse cortical neurons induces the expression of FMRP and FMRP‐dependent ER stress response and subsequent translational suppression. Also Aβ induces FMRP‐dependent neuronal hyposynchronicity . We further characterized that two translational regulators, eukaryotic translation initiation factor 2α (eIF2α) and eukaryotic translation elongation factor 2 (eEF2), are involved in Aβ induced translational suppression and are dysregulated in the absence of FMRP. Mechanistically we found that FMRP is responsible for regulating the levels of Protein Phosphatase 2 (PP2A) and Protein Phosphatase 1 (PP1) that differentially modulate the phosphorylation status and thereby activity of eIF2α and eEF2 following treatment of Aβ. Using a pharmacological approach to inhibit PP2A and PP1 with Okadaic Acid, we were able to rescue the impairment of eIF2α and eEF2 as well as the translational suppression and lack of neuronal hyposynchrony in the absence of FMRP.ConclusionThese results suggest a novel function of FMRP in regulating translational suppression and neuronal networking function following Aβ treatment by fine‐tuning two distinct signaling pathways involved in global translation.

Tumor Treating Fields (TTFields) Concomitant with Immune Checkpoint Inhibitors Are Therapeutically Effective in Non-Small Cell Lung Cancer (NSCLC) In Vivo Model.

Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields induce anti-mitotic effects through the disruption of the mitotic spindle and abnormal chromosome segregation, which trigger several forms of cell death, including immunogenic cell death (ICD). The efficacy of TTFields concomitant with anti-programmed death-1 (anti-PD-1) treatment was previously shown in vivo and is currently under clinical investigation. Here, the potential of TTFields concomitant with anti- PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed death-ligand 1 (anti-PD-L1) immune checkpoint inhibitors (ICI) to improve therapeutic efficacy was examined in lung tumor-bearing mice. Increased circulating levels of high mobility group box 1 protein (HMGB1) and elevated intratumoral levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) were found in the TTFields-treated mice, indicative of ICD induction. The concomitant application of TTFields and ICI led to a significant decrease in tumor volume as compared to all other groups. In addition, significant increases in the number of tumor-infiltrating immune cells, specifically cytotoxic T-cells, were observed in the TTFields plus anti-PD-1/anti-CTLA-4 or anti-PD-L1 groups. Correspondingly, cytotoxic T-cells isolated from these tumors showed higher levels of IFN-γ production. Collectively, these results suggest that TTFields have an immunoactivating role that may be leveraged for concomitant treatment with ICI to achieve better tumor control by enhancing antitumor immunity.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Recovery Sleep Immediately after Prolonged Sleep Deprivation Stimulates the Transcription of Integrated Stress Response-Related Genes in the Liver of Male Rats.

Sleep loss induces performance impairment and fatigue. The reactivation of human herpesvirus-6, which is related to the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), is one candidate for use as an objective biomarker of fatigue. Phosphorylated eIF2α is a key regulator in integrated stress response (ISR), an intracellular stress response system. However, the relation between sleep/sleep loss and ISR is unclear. The purpose of the current study was to evaluate the effect of prolonged sleep deprivation and recovery sleep on ISR-related gene expression in rat liver. Eight-week-old male Sprague-Dawley rats were subjected to a 96-hour sleep deprivation using a flowerpot technique. The rats were sacrificed, and the liver was collected immediately or 6 or 72 h after the end of the sleep deprivation. RT-qPCR was used to analyze the expression levels of ISR-related gene transcripts in the rat liver. The transcript levels of the Atf3, Ddit3, Hmox-1, and Ppp15a1r genes were markedly increased early in the recovery sleep period after the termination of sleep deprivation. These results indicate that both activation and inactivation of ISRs in the rat liver occur simultaneously in the early phase of recovery sleep.

The Influence of Supplemental Zinc and Ractopamine Hydrochloride on the Performance and Longissimus Thoracis Proteome of Finishing Beef Steers

To determine how Zn and ractopamine hydrochloride (RAC) supplementation affect the longissimus thoracis muscle proteome in beef cattle, 48 high percentage Angus steers (494±18.2 kg) were utilized in a 2×2 factorial study design. Steers were blocked by body weight (BW) and genetic gain potential (GeneMax; Zoetis, Parsippany, NJ) into control (CON; analyzed 36 mg Zn/kg dry matter [DM]) or supranutritional Zn (SUPZN; CON+60 mg Zn/kg DM from ZnSO4+60 mg Zn/kg DM from Zn-amino acid complex; Availa Zn, Zinpro, Eden Prairie, MN) dietary treatments (ZNTRT). Starting 28 d prior to harvest, steers were blocked by BW within ZNTRT to RAC treatments (RACTRT) of 0 (NO) or 300 mg·steer−1·d−1 (RAC; Actogain 45, Zoetis). After 15 d on RACTRT, longissimus thoracis biopsies were collected from all steers for proteomic analysis. Twenty-eight steers (n=7 per treatment) were harvested at a commercial abattoir on Day 90. A ZNTRT×RACTRT effect showed SUPZN-RAC steers had a greater dressing percentage than other treatments (P≤0.02). Steers fed SUPZN had a greater hot carcass weight, carcass-adjusted final BW, overall average daily gain, and overall gain to feed (P≤0.05). Differentially abundant proteins involved in energy metabolism, muscle structure, and protein synthesis potentially indicate muscle fiber characteristic differences because of Zn and RAC supplementation.Both myosin light chain kinase 2 and eukaryotic translation initiation factor 5A-1 were more abundant in SUPZN-RAC steers than SUPZN-NO (P≤0.05) or CON-NO (P≤0.04) steers when analyzed for phosphorylation modifications. L-lactate dehydrogenase B was more abundant in SUPZN-NO steers than CON-NO (P=0.03) or CON-RAC (P=0.01) steers. In conclusion, increased Zn supplementation may be needed to optimize the hypertrophic effects of RAC through its effects on the longissimus thoracis proteome.

Anticancer effects and mechanisms of astragaloside‑IV (Review).

Astragalusmembranaceus Bunge is widely used in Traditional Chinese Medicine to treat various cancers. Astragaloside‑IV (AS‑IV) is one of the major compounds isolated from A.membranaceus Bunge and has been demonstrated to have antitumor effects by inhibiting cell proliferation, invasion and metastasis in various cancer types. Numerous studies have used invitro cell culture and invivo animal models of cancer to explore the antitumor activities of AS‑IV. In the present study, the antitumor effects and mechanisms of AS‑IV reported in studies recorded in the PubMed database were reviewed. First, the antitumor effects of AS‑IV on proliferation, cell cycle, apoptosis, autophagy, invasion, migration, metastasis and epithelial‑mesenchymal transition processes in cancer cells and the tumor microenvironment, including angiogenesis, tumor immunity and macrophage‑related immune responses to cancer cells, were comprehensively discussed. Subsequently, the molecular mechanisms and related signaling pathways associated with antitumor effects of AS‑IV as indicated by invitro and invivo studies were summarized, including the Wnt/AKT/GSK-3β (glycogen synthase kinase‑3β)/β‑catenin, TGF‑β/PI3K/AKT/mTOR, PI3K/MAPK/mTOR, PI3K/AKT/NF‑κB, Rac family small GTPase 1/RAS/MAPK/ERK, TNF‑α/protein kinaseC/ERK1/2‑NF‑κB and Tregs (T‑regulatory cells)/IL‑11/STAT3 signaling pathways. Of note, several novel mechanisms of Toll‑like receptor 4 (TLR4)/NF‑κB/STAT3, pSmad3C/3L, nuclear factor erythroid 2‑related factor (NrF2)/heme oxygenase1, circDLST/microRNA‑489‑3p/eukaryotic translation initiation factor 4A1 and macrophage‑related high‑mobility group box 1‑TLR4 signaling pathways associated with the anticancer activity of AS‑IV were also included. Finally, the limitations of current studies that must be addressed in future studies were pointed out to facilitate the establishment of AS‑IV as a potent therapeutic drug in cancer treatment.