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Background Large-scale production and application of amorphous silica nanoparticles (SiNPs) have enhanced the risk of human exposure to SiNPs. However, the toxic effects and the underlying biological mechanisms of SiNPs on Caenorhabditis elegans remain largely unclear. Purpose This study was to investigate the genome-wide transcriptional alteration of SiNPs on C. elegans. Methods and results In this study, a total number of 3105 differentially expressed genes were identified in C. elegans. Among them, 1398 genes were significantly upregulated and 1707 genes were notably downregulated in C. elegans. Gene ontology analysis revealed that the significant change of gene functional categories triggered by SiNPs was focused on locomotion, determination of adult lifespan, reproduction, body morphogenesis, multicellular organism development, endoplasmic reticulum unfolded protein response, oocyte development, and nematode larval development. Meanwhile, we explored the regulated effects between microRNA and genes or signaling pathways. Pathway enrichment analysis and miRNA-gene-pathway-network displayed that 23 differential expression microRNA including cel-miR-85-3p, cel-miR-793, cel-miR-241-5p, and cel-miR-5549-5p could regulate the longevity-related pathways and inflammation signaling pathways, etc. Additionally, our data confirmed that SiNPs could disrupt the locomotion behavior and reduce the longevity by activating ins-7, daf-16, ftt-2, fat-5, and rho-1 genes in C. elegans. Conclusion Our study showed that SiNPs induced the change of the whole transcriptome in C. elegans, and triggered negative effects on longevity, development, reproduction, and body morphogenesis. These data provide abundant clues to understand the molecular mechanisms of SiNPs in C. elegans.

Lipofectamine 2000 (Lipo2000) delivery system is commonly used for short interfering RNA (siRNA) transfection, whereas the cellular responses have attracted little attention. The purpose of this study is to evaluate the effect of siRNA transfection using Lipo2000 on cellular functions and the possible underlying mechanism. Primary human umbilical vein endothelial cells (HUVECs) and adult human coronary artery endothelial cell line (HCAECs) were treated with different concentrations of a Lipo2000/negative control siRNA (NC siRNA) complex or Lipo2000 for specific durations. The cell proliferation, apoptosis rate, and protein expression of claudin5 (CLDN5) and ETS-related gene (ERG) were analyzed as indicators of cellular function. The effects of the Lipo2000/NC siRNA complex on cellular autophagy and endoplasmic reticulum (ER) unfolded protein response (UPR) were investigated by western blot and real-time polymerase chain reaction analyses; autophagy was also evaluated by transmission electron microscopy. The Lipo2000/NC siRNA complex inhibited proliferation, downregulated various proteins, and increased the apoptosis in both HUVECs and HCAECs. Both autophagy and UPR were observed in HUVECs treated with the Lipo2000/NC siRNA complex, ER stress-induced autophagy acted as a cellular protective factor against apoptosis, as inhibition of autophagy by chemical inhibitors increased the cell apoptosis rate. Chemical chaperones failed to prevent the Lipo2000/siRNA complex-induced UPR. However, knockdown of protein kinase RNA-like ER kinase and inositol-requiring protein 1, instead of activating transcription factor-6, partially ameliorated the UPR and reversed the protein level of CLDN5 and ERG downregulated by Lipo2000/NC siRNA complex. This study provides the first evidence that the Lipo2000-mediated transport of siRNA leads to an increase in UPR and ER stress-related apoptosis in endothelial cells.

Breast cancer (BC) is the second most frequent cause of death among women. Representing a complex and heterogeneous type of cancer, its occurrence is attributed by both genetic (gene mutations, e.g., BRCA1, BRCA2) and non-genetic (race, ethnicity, etc.) risk factors. The effectiveness of available treatment regimens (small molecules, cytotoxic agents, and inhibitors) decreased due to their poor penetration across biological barriers, limited targeting, and rapid body clearance along with their effect on normal resident cells of bone marrow, gastrointestinal tract, and hair follicles. This significantly reduced their clinical outcomes, which led to an unprecedented increase in the number of cases worldwide. Nanomedicine, a nano-formulation of therapeutics, emerged as a versatile delivering module for employment in achieving the effective and target specific delivery of pharmaceutical payloads. Adoption of nanotechnological approaches in delivering therapeutic molecules to target cells ensures not only reduced immune response and toxicity, but increases the stability of therapeutic entities in the systemic circulation that averts their degradation and as such increased extravasations and accumulation via enhanced permeation and the retention (EPR) effect in target tissues. Additionally, nanoparticle (NP)-induced ER stress, which enhances apoptosis and autophagy, has been utilized as a combative strategy in the treatment of cancerous cells. As nanoparticles-based avenues have been capitalized to achieve better efficacy of the new genera of therapeutics with enhanced specificity and safety, the present study is aimed at providing the fundamentals of BC, nanotechnological modules (organic, inorganic, and hybrid) employed in delivering different therapeutic molecules, and mechanistic insights of nano-ER stress induced apoptosis and autophagy with a perspective of exploring this avenue for use in the nano-toxicological studies. Furthermore, the current scenario of USA FDA approved nano-formulations and the future perspective of nanotechnological based interventions to overcome the existing challenges are also discussed.

Viruses elicit cell and organismic stress, and offset homeostasis. They trigger intrinsic, innate and adaptive immune responses, which limit infection. Viruses restore homeostasis by harnessing evolutionary conserved stress responses, such as the endoplasmic reticulum (ER) unfolded protein response (UPRER). The canonical UPRER restores homeostasis based on a cell-autonomous signalling network modulating transcriptional and translational output. The UPRER remedies cell damage, but upon severe and chronic stress leads to cell death. Signals from the UPRER flow along three branches with distinct stress sensors, the inositol requiring enzyme (Ire) 1, protein kinase R (PKR)-like ER kinase (PERK), and the activating transcription factor 6 (ATF6). This review shows how both enveloped and non-enveloped viruses use the UPRER to control cell stress and metabolic pathways, and thereby enhance infection and progeny formation, or undergo cell death. We highlight how the Ire1 axis bypasses apoptosis, boosts viral transcription and maintains dormant viral genomes during latency and persistence periods concurrent with long term survival of infected cells. These considerations open new options for oncolytic virus therapies against cancer cells where the UPRER is frequently upregulated. We conclude with a discussion of the evolutionary impact that viruses, in particular retroviruses, and anti-viral defense has on the UPRER., Although viruses trigger robust stress responses and innate and adaptive host defense, we reveal that a large spectrum of enveloped and non-enveloped viruses activate homeostasis restoring processes, especially the endoplasmic reticulum unfolded protein response, which promotes virus propagation and survival.

Bats hibernate to survive stressful conditions. Examination of whole cell and mitochondrial proteomes of the liver of Myotis ricketti revealed that torpid bats had endoplasmic reticulum unfolded protein response (UPRER), global reduction in glycolysis, enhancement of lipolysis, and selective amino acid metabolism. Compared to active bats, torpid bats had higher amounts of phosphorylated serine/threonine kinase (p-Akt) and UPRER markers such as PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 4 (ATF4). Torpid bats also had lower amounts of the complex of Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (p65)/I-κBα. Cellular redistribution of 78 kDa glucose-regulated protein (GRP78) and reduced binding between PERK and GRP78 were also seen in torpid bats. Evidence of such was not observed in fasted, cold-treated, or normal mice. These data indicated that bats activate Akt, Nrf2, and NF-κB via the PERK-ATF4 regulatory axis against endoplasmic reticulum stresses during hibernation., Huang et al. use proteomics to examine liver and liver mitochondria of hibernating bats. The authors observe the unfolded protein response in endoplasmic reticulum and co-activation of survival signaling pathways in torpid bats and provide further insight into physiological adaptations that allow bats to survive a long period of hibernation.

Background and Objective: Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. Mesenchymal stem cells (MSCs) regulate bone mass homeostasis in AIS, which might be related to the pathogenesis of AIS. However, the mRNA–miRNA–lncRNA network linked to the regulation of the genetic pathogenesis of MSCs remains unknown. Methods: We conducted an exhaustive literature search of PubMed, EMBASE, and the Gene Expression Omnibus database to find differentially expressed genes (DEGs), differentially expressed miRNAs (DE miRNAs), and differentially expressed lncRNAs (DE lncRNAs). Functional enrichment analysis was performed through Enrichr database. Protein–protein interaction (PPI) network was constructed using STRING database, and hub genes were identified by CytoHubba. Potential regulatory miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and RNA22, respectively. Results: We identified 551 upregulated and 476 downregulated genes, 42 upregulated and 12 downregulated miRNAs, and 345 upregulated and 313 downregulated lncRNAs as DEGs, DE miRNAs, and DE lncRNAs, respectively. Functional enrichment analysis revealed that they were significantly enriched in protein deglutamylation and regulation of endoplasmic reticulum unfolded protein response. According to node degree, one upregulated hub gene and eight downregulated hub genes were identified. After drawing the Venn diagrams and matching to Cytoscape, an mRNA–miRNA–lncRNA network linked to the pathogenesis of MSCs in AIS was constructed. Conclusion: We established a novel triple regulatory network of mRNA–miRNA–lncRNA ceRNA, among which all RNAs may be utilized as the pathogenesis biomarker of MSCs in AIS.

Background: In human tauopathies, pathological aggregation of misfolded/unfolded proteins, particularly microtubule-associated protein tau (MAPT, tau) is considered to be an essential mechanism that triggers the induction of endoplasmic reticulum (ER) stress. Objective: Here, we assessed the molecular effects of natural antioxidant alpha-lipoic acid (ALA) in human tauR406W (hTau)-induced ER unfolded protein response (ERUPR) in fruit flies. Methods: In order to reduce hTau neurotoxicity during brain development, we used a transgenic model of tauopathy where the maximum toxicity was observed in adult flies. Then, the effects of ALA (0.001, 0.005, and 0.025% w/w of diet) in htau-induced ERUPR and behavioral dysfunctions in the ages 20 and 30 days were evaluated in Drosophila melanogaster. Results: Data from expression (mRNA and protein) patterns of htau, analysis of eyes external morphology as well as larvae olfactory memory were confirmed by our tauopathy model. Moreover, the expression of ERUPR-related proteins involving Activating Transcription Factor 6 (ATF6), inositol regulating enzyme 1 (IRE1), and protein kinase RNA-like ER kinase (PERK) wase upregulated and locomotor function decreased in both ages of the model flies. Remarkably, the lower dose of ALA modified ERUPR and supported the reduction of behavioral deficits in youngest adults through the enhancement of GRP87/Bip, reduction of ATF6, downregulation of PERK-ATF4 pathway, and activation of the IRE1-XBP1 pathway. On the other hand, only a higher dose of ALA affected the ERUPR via moderation of PERK-ATF4 signaling in the oldest adults. As ALA also exerts higher protective effects on the locomotor function of younger adults when htauR406Wis expressed in all neurons (htau-elav) and mushroom body neurons (htau-ok), we proposed that ALA has age-dependent effects in this model. Conclusion: Taken together, based on our results, we conclude that aging potentially influences the ALA effective dose and mechanism of action on tau-induced ERUPR. Further molecular studies will warrant possible therapeutic applications of ALA in age-related tauopathies.

BackgroundIn human tauopathies, pathological aggregation of misfolded/unfolded proteins particularly microtubule-associated protein tau (MAPT, tau) is considered to be essential mechanisms that trigger the induction of endoplasmic reticulum (ER) stress. Here we assessed the molecular effects of natural antioxidant alpha-lipoic acid (ALA) in human tauR406W (htau)-induced ER unfolded protein response (ERUPR) in the young and older flies.MethodsIn order to reduce htau neurotoxicity during brain development, we used a transgenic model of tauopathy where the maximum toxicity was observed in adult flies. Then, the effects of ALA (0.001, 0.005, and 0.025% w/w of diet) in htau-induced ERUPR in the ages 20 and 30 days were evaluated.ResultsData from expression (mRNA and protein) patterns of htau, analysis of eyes external morphology as well as larvae olfactory memory were confirmed our tauopathy model. Moreover, expression of ERUPR-related proteins involving activating transcription factor 6 (ATF6), inositol regulating enzyme 1 (IRE1), and protein kinase RNA-like ER kinase (PERK) were upregulated and locomotor function decreased in both ages of the model flies. Remarkably, the lower dose of ALA modified ERUPR and supported the reduction of behavioral deficits in youngest adults through enhancement of GRP87/Bip, reduction of ATF6, downregulation of PERK-ATF4 pathway, and activation of the IRE1-XBP1 pathway. On the other hand, only a higher dose of ALA was able to affect the ERUPR via moderation of PERK-ATF4 signaling in the oldest adults. As ALA exerts their higher protective effects on the locomotor function of younger adults when htauR406W expressed in all neurons (htau-elav) and mushroom body neurons (htau-ok), we proposed that ALA has age-dependent effects in this model.ConclusionTaken together, based on our results we conclude that aging potentially influences the ALA effective dose and mechanism of action on tau-induced ERUPR. Further molecular studies will warrant possible therapeutic applications of ALA in age-related tauopathies.

Background Chronic kidney disease (CKD) is a worldwide public health problem due to its increasing prevalence worldwide. Endoplasmic reticulum (ER) stress has been shown to play a role in the pathogenesis of various renal diseases in humans. It leads to the activation of the unfolded protein response (UPR) which triggers three known trans membrane sensors in the ER: activating transcription factor 6 (ATF6), inositol-requiring enzyme I (IRE1), and PKR (double-stranded RNA-dependent protein kinase)-like ER protein kinase (PERK). The activation of these signal transduction pathways can result in cell death, inflammation, and fibrosis in the context of CKD. Objectives The aim of this study was to detect the level of gene expression of activating transcription factor 6 (ATF6), inositol-requiring enzyme I (IRE1), and PKR (double-stranded RNA-dependent protein kinase)-like ER protein kinase (PERK) in chronic kidney disease patients. Subjects and methods This study was carried out on eighty subjects, 50 patients with CKD (25 with hypertension and 25 without hypertension) and 30 healthy subjects served as controls. All studied subjects underwent laboratory investigations, including CBC, Serum Lipid profile: Total cholesterol, Triglycerides, HDL-cholesterol and LDL-cholesterol, liver and kidney functions, fasting and 2 h postprandial blood glucose and HbA1C, serum level of IL6 and gene expression of ATF6, IRE1 and PERK using real time PCR technique. Results There was a significant increase in relative quantitation (RQ) of gene expression of IRE1, ATF6 and PERK in chronic kidney patient groups with hypertension and without hypertension compared to control group. Also, there was a significant positive correlation of PERK and ATF6 gene expressions and a significant negative correlation of PERK gene expressions and GFR in groups I&II. Conclusion Endoplasmic reticulum (ER) stress occurs in CKD with activation of gene expression of three trans-membrane sensors in the ER: activating transcription factor 6 (ATF6), inositol-requiring enzyme I (IRE1), and PKR (double-stranded RNA-dependent protein kinase)-like ER protein kinase (PERK)., Highlights • Endoplasmic reticulum (ER) stress occurs in CKD. • A significant increase in gene expression of PERK, ATF6 and IRE1 was detected in chronic kidney patients. • Patients with CKD have a higher prevalence of dyslipidemias. • Serum pro-inflammatory cytokines (IL-6) may be predictive of the development of. • CKD patients should be considered as patients at very high risk for CVD.

Idiopathic pulmonary fibrosis (IPF) is a group of chronic interstitial pulmonary diseases characterized by myofibroblast proliferation and extracellular matrix deposition with limited treatment options. Based on our previous observation, we hypothesized microcystin-leucine arginine (LR), an environmental cyanobacterial toxin, could potentially suppress pulmonary fibrosis. In this study, we first demonstrated that chronic exposure of microcystin-LR by oral for weeks indeed attenuated the pulmonary fibrosis both on bleomycin-induced rat and fluorescein isothiocyanate-induced mouse models. Our data further indicated that treatment with microcystin-LR substantially reduced TGF-β1/Smad signaling in rat pulmonary tissues. The experiments in vitro found that microcystin-LR was capable of blocking epithelial–mesenchymal transition (EMT) and fibroblast–myofibroblast transition (FMT) through suppressing the differentiation of CD206+ macrophages. Mechanically, microcystin-LR was found to bind to glucose-regulated protein 78 kDa (GRP78) and suppress endoplasmic reticulum unfolded protein response (UPRER) signaling pathways. These events led to the modulation of M2 polarization of macrophages, which eventually contributed to the alleviation of pulmonary fibrosis. Our results revealed a novel mechanism that may account for therapeutic effect of microcystin-LR on IPF.

As a vital member of AAA+ (ATPase associated with diverse cellular activities) protein superfamily, Lon, a homo-hexameric ring-shaped protein complex with a serine-lysine catalytic dyad, is highly conserved throughout almost all prokaryotic and eukaryotic organisms. Lon protease (LONP) plays an important role in maintaining mitoproteostasis through selectively recognizing and degrading oxidatively modified mitoproteins within mitochondrial matrix, such as oxidized aconitase, phosphorylated mitochondrial transcription factor A, etc. Furthermore, the up-regulated LONP increased mitochondrial ROS generation to promote cell survival, cell proliferation, epithelial-mesenchymal transition, and cell migration, which was attributed to the up-regulation of NADH:ubiquinone oxidoreductase core subunit S8 via interaction with chaperone Lon under hypoxic or oxidative stress in tumorigenesis. In addition, Lon also participated in protein kinase RNA (PKR)-like endoplasmic reticulum kinase signaling pathway under endoplasmic reticulum (ER) stress. In short, Lon, as a pivotal stress-responsive protein that involved in the crosstalks among mitochondria, ER and nucleus, participated in multifarious important cellular processes crucial for cell survival, such as the mitochondrial protein quality control system, the mitochondrial unfolded protein response, the mtDNA maintenance, and the ER unfolded protein response.

The benefits of exercise on the heart are well recognized, and clinical studies have demonstrated that exercise is an intervention that can improve cardiac function in heart failure patients. This has led to significant research into understanding the key mechanisms responsible for exercise-induced cardiac protection. Here, we summarize molecular mechanisms that regulate exercise-induced cardiac myocyte growth and proliferation. We discuss in detail the effects of exercise on other cardiac cells, organelles, and systems that have received less or little attention and require further investigation. This includes cardiac excitation and contraction, mitochondrial adaptations, cellular stress responses to promote survival (heat shock response, ubiquitin-proteasome system, autophagy-lysosomal system, endoplasmic reticulum unfolded protein response, DNA damage response), extracellular matrix, inflammatory response, and organ-to-organ crosstalk. We summarize therapeutic strategies targeting known regulators of exercise-induced protection and the challenges translating findings from bench to bedside. We conclude that technological advancements that allow for in-depth profiling of the genome, transcriptome, proteome and metabolome, combined with animal and human studies, provide new opportunities for comprehensively defining the signaling and regulatory aspects of cell/organelle functions that underpin the protective properties of exercise. This is likely to lead to the identification of novel biomarkers and therapeutic targets for heart disease.

Trachemys scripta elegans, as a freshwater invasive species, can survive and lay eggs in brackish water, which may lead to the expansion of its potential invasion range due to freshwater salinization. Our previous studies have shown that high salinity leads to the accumulation of serum lipid content, which may induce endoplasmic reticulum stress (ERS) in the turtle. To better understand whether ERS is triggered by salinity, and in turn whether the turtles promote the protection mechanism, we exposed the turtles to the freshwater (CK), 5‰ salinity water (S5) and 15‰ salinity water (S15), and sampled at 6 h, 24 h and 30 d. 13 differentially expressed genes (DEGs) related to ERS pathways were found in the comparison of CK vs. S15 by transcriptomics analysis. Then, the mRNA and protein expression of ERS and its related activation pathways were further investigated. ERS marker glucose regulated protein 78 kD (GRP78) increased significantly (p 0.05) in both the transcript and protein levels after exposure to 15‰ salinity water, which clearly indicated that salinity could induce ERS in T. s. elegans. Meanwhile, the three unfolded protein response (UPR) including transducers protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor-6 (ATF6) were promoted by salinity, suggesting that the turtle might promote physiological process to eliminate damaged cells and cope with unfolded proteins accumulation induced by ERS. Our results provide new insight into the mechanism of salinity adaptation in T. s. elegans and salt-tolerant biological invasion.

Nanoplastics can be used in various fields, such as personal care products. Nevertheless, the effect of nanoplastic exposure on metabolism and its association with stress response remain largely unclear. Using Caenorhabditis elegans as an animal model, we determined the effect of nanopolystyrene exposure on lipid metabolism and its association with the response to nanopolystyrene. Exposure (from L1-larave to adult day-3) to 100 nm nanopolystyrene (≥1 μg/L) induced severe lipid accumulation and increase in expressions of mdt-15 and sbp-1 encoding two lipid metabolic sensors. Meanwhile, we found that SBP-1 acted downstream of intestinal MDT-15 during the control of response to nanopolystyrene. Intestinal transcriptional factor SBP-1 activated two downstream targets, fatty acyl CoA desaturase FAT-6 and heat-shock protein HSP-4 (a marker of endoplasmic reticulum unfolded protein response (ER UPR)) to regulate nanopolystyrene toxicity. Both MDT-15 and SBP-1 were involved in the activation of ER-UPR in nanopolystyrene exposed nematodes. Moreover, SBP-1 regulated the innate immune response by activating FAT-6 in nanopolystyrene exposed nematodes. In the intestine, function of MDT-15 and SBP-1 in regulating nanopolystyrene toxicity was under the control of upstream signaling cascade (PMK-1-SKN-1) in p38 MAPK signaling pathway. Therefore, our data raised an important molecular basis for potential protective function of lipid metabolic response in nanopolystyrene exposed nematodes.

10 p. Aging has been related with a decline in the ability to handle protein folding, which leads to endoplasmic reticulum stress and alterations in unfolded protein response (UPR). Importantly, physical activity could activate the UPR and attenuate or prevent age-induced endoplasmic reticulum (ER) dysfunction. The current study evaluated the effects of a resistance exercise on UPR and mitochondrial functions in peripheral blood mononuclear cells (PBMCs) from elderly subjects. Thirty healthy women and men (age, 72.8, sx− = 2.2 years) were randomized to a training group, which performed an 8-week resistance training programme, or a control group, which followed their daily routines. The phosphorylation of PERK and IRE1, as well as ATF4, and XBP1 protein expression, significantly increased following the training, while expression of BiP, AFT6 and CHOP remain without changes. Additionally, the intervention also induced an increase in PGC-1α and Mfn1 protein levels, while no changes were found in Drp1 expression. Finally, the resistance protocol was not able to activate PINK1/Parkin and Bnip3/Nix pathways. The results obtained seem to indicate that 8-week resistance exercise activates the UPR, stimulates mitochondrial biogenesis, maintains mitochondrial dynamics and prevents mitophagy activation by unfolded proteins in PBMCs from elderly subjects. SI

Background: Idiopathic pulmonary fibrosis (IPF) refers to a group of chronic and untreatable fatal interstitial pulmonary diseases characterized by myofibroblast proliferation and extracellular matrix (ECM) deposition. The pathogenesis of IPF involves alternative activation of macrophages and excessive transforming growth factor β1 (TGF-β1) signaling. Microcystin-leucine arginine (microcystin-LR) is an environmental toxin produced by cyanobacteria. We previously observed a downregulated TGF-β1 expression in pulmonary tissues as a result of chronic exposure to microcystin-LR, which prompted us to assume a therapeutic role of this molecule on IPF. Methods: The therapeutic effect of microcystin-LR was evaluated using a rat model of bleomycin-induced pulmonary fibrosis and the confirmation test was performed with a fluorescein isothiocyanate-induced mouse pulmonary fibrosis model. An in vitro coculture model of monocytes/macrophages with alveolar epithelial cells or with fibroblasts was established to investigate the mechanisms of microcystin-LR-mediated anti-fibrotic effect. Findings: Treatment with microcystin-LR substantially reduced TGF-β1/Smad signaling and obviously alleviated the lesion of pulmonary fibrosis in animal models. Microcystin-LR was capable of blocking epithelialmesenchymal transition and fibroblast-myofibroblast transition through suppressing the differentiation of CD206+ macrophages. Furthermore, microcystin-LR could bind to glucose-regulated protein 78 kDa (GRP78) and suppress endoplasmic reticulum unfolded protein response signaling pathways. Moreover, no additional lesion related with the regimen of microcystin-LR was observed on the animal models in this study. Interpretation: We have provided evidences demonstrating that microcystin-LR could alleviate pulmonary fibrosis by antagonizing macrophage polarization to an M2-like phenotype. Our data revealed a novel mechanism that may account for therapeutic effect of microcystin-LR on IPF. Funding Statement: This work was supported by the National Natural Science Foundation of China (81270152, 81771504 and 81501977), Human Resource Summit Grant of Jiangsu Province (WSN-043) and The Young Talents Program of Jiangsu Cancer Hospital (QL201807). Declaration of Interests: All of the authors declared that no conflict of interest exists. Ethics Approval Statement: The animal care and the study procedures were approved by the Ethics Committee for Animal Research in Medical School of Nanjing University.

In this work, we examine the relationship between stress resistance and aging. We find that resistance to multiple types of stress peaks during early adulthood and then declines with age. To dissect the underlying mechanisms, we use C. elegans transcriptional reporter strains that measure the activation of different stress responses including: the heat shock response, mitochondrial unfolded protein response, endoplasmic reticulum unfolded protein response, hypoxia response, SKN-1-mediated oxidative stress response, and the DAF-16-mediated stress response. We find that the decline in stress resistance with age is at least partially due to a decreased ability to activate protective mechanisms in response to stress. In contrast, we find that any baseline increase in stress caused by the advancing age is too mild to detectably upregulate any of the stress response pathways. Further exploration of how worms respond to stress with increasing age revealed that the ability to mount a hormetic response to heat stress is also lost with increasing age. Overall, this work demonstrates that resistance to all types of stress declines with age. Based on our data, we speculate that the decrease in stress resistance with advancing age results from a genetically-programmed inactivation of stress response pathways, not accumulation of damage.

Nanopolystyrene particles have been widely used in many fields. However, the molecular responses of organisms to nanopolystyrene particles at predicted environmentally relevant concentrations are still largely unclear. Caenorhabditis elegans is employed herein to investigate the molecular response of p38 mitogen-activated protein kinase (MAPK) signaling to nanoplastic particles (1 µg L-1 ) and the underlying molecular mechanism. In wild-type nematodes, prolonged exposure (from L1-larvae to adult day 3) to nanopolystyrene particles increases the expression of pmk-1 encoding a p38 MAPK. Mutation of pmk-1 induces a susceptibility to nanopolystyrene toxicity, suggesting that the p38 MAPK signaling mediates a protective response to nanopolystyrene particles. PMK-1 functions in the intestine to act upstream of two transcriptional factors (ATF-7 and SKN-1), which can further act upstream of XBP-1, a key regulator of endoplasmic reticulum unfolded protein response (ER UPR), to regulate the response to nanopolystyrene particles. PMK-1, ATF-7, SKN-1, and XBP-1 are all required for the induction of intestinal ER UPR in nematodes exposed to nanopolystyrene particles. Therefore, the intestinal p38 MAPK signaling may mediate a protective response to nanopolystyrene particles by activating XBP-1-mediated ER UPR. The results herein highlight the importance of molecular response in the intestine of organisms to nanopolystyrene particles.

9 p. The endoplasmic reticulum (ER) is a dynamic and multifunctional organelle responsible for protein biosynthesis, folding, assembly and modifications. Loss of protein folding regulation, which leads to unfolded or misfolded proteins accumulation inside the ER lumen, drives ER stress (ERS) and unfolded protein response (UPR) activation. During aging, there is a decline in the ability of the cell to handle protein folding, accumulation and aggregation, and the function of UPR is compromised. There is a progressive failure of the chaperoning systems and a decline in many of its components, so that the UPR activation cannot rescue the ERS. Physical activity has been proposed as a powerful tool against aged-related diseases, which are linked to ERS. Interventional studies have demonstrated that regular exercise is able to decrease oxidative stress and inflammation and reverse mitochondrial and ER dysfunctions. Exercise-induced metabolic stress could activate the UPR since muscle contraction is directly involved in its activation, mediating exercise-induced adaptation responses. In fact, regular moderate-intensity exercise-induced ERS acts as a protective mechanism against current and future stressors. However, biological responses vary according to exercise intensity and therefore induce different degrees of ERS and UPR activation. This article reviews the effects of aging and exercise on ERS and UPR, also analyzing possible changes induced by different types of exercise in elderly subjects. SI

Alzheimer’s disease (AD), a neurodegenerative disorder, is most common cause of dementia witnessed among aged people. The pathophysiology of AD develops as a consequence of neurofibrillary tangle formation which consists of hyperphosphorylated microtubule associated tau protein and senile plaques of amyloid-β (Aβ) peptide in specific brain regions that result in synaptic loss and neuronal death. The feeble buffering capacity of endoplasmic reticulum (ER) proteostasis in AD is evident through alteration in unfolded protein response (UPR), where UPR markers express invariably in AD patient’s brain samples. Aging weakens UPRER causing neuropathology and memory loss in AD. This review highlights molecular signatures of UPRER and its key molecular alliance that are affected in aging leading to the development of intriguing neuropathologies in AD. We present a summary of recent studies reporting usage of small molecules as inhibitors or activators of UPRER sensors/effectors in AD that showcase avenues for therapeutic interventions.

Liver cancer is a serious threat to public health and has fairly complicated pathogenesis. Therefore, the identification of key genes and pathways is of much importance for clarifying molecular mechanism of hepatocellular carcinoma (HCC) initiation and progression. HCC-associated gene expression dataset was downloaded from Gene Expression Omnibus database. Statistical software R was used for significance analysis of differentially expressed genes (DEGs) between liver cancer samples and normal samples. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, based on R software, were applied for the identification of pathways in which DEGs significantly enriched. Cytoscape software was for the construction of protein–protein interaction (PPI) network and module analysis to find the hub genes and key pathways. Finally, weighted correlation network analysis (WGCNA) was conducted to further screen critical gene modules with similar expression pattern and explore their biological significance. Significance analysis identified 1230 DEGs with fold change >2, including 632 significantly down-regulated DEGs and 598 significantly up-regulated DEGs. GO term enrichment analysis suggested that up-regulated DEG significantly enriched in immune response, cell adhesion, cell migration, type I interferon signaling pathway, and cell proliferation, and the down-regulated DEG mainly enriched in response to endoplasmic reticulum stress and endoplasmic reticulum unfolded protein response. KEGG pathway analysis found DEGs significantly enriched in five pathways including complement and coagulation cascades, focal adhesion, ECM–receptor interaction, antigen processing and presentation, and protein processing in endoplasmic reticulum. The top 10 hub genes in HCC were separately GMPS, ACACA, ALB, TGFB1, KRAS, ERBB2, BCL2, EGFR, STAT3, and CD8A, which resulted from PPI network. The top 3 gene interaction modules in PPI network enriched in immune response, organ development, and response to other organism, respectively. WGCNA revealed that the confirmed eight gene modules significantly enriched in monooxygenase and oxidoreductase activity, response to endoplasmic reticulum stress, type I interferon signaling pathway, processing, presentation and binding of peptide antigen, cellular response to cadmium and zinc ion, cell locomotion and differentiation, ribonucleoprotein complex and RNA processing, and immune system process, respectively. In conclusion, we identified some key genes and pathways closely related with HCC initiation and progression by a series of bioinformatics analysis on DEGs. These screened genes and pathways provided for a more detailed molecular mechanism underlying HCC occurrence and progression, holding promise for acting as biomarkers and potential therapeutic targets.

Enteroendocrine cells (EEC) have been studied extensively for their ability to regulate gastrointestinal motility and insulin release by secretion of peptide hormones. In particular, the L cell-derived incretin glucagon-like peptide 1 has gained enormous attention due to its insulinotropic action and relevance in the treatment of type 2 diabetes. Yet, accumulating data indicates a critical role for EEC and incretins in metabolic adaptation and in orchestrating immune responses beyond blood glucose control. EEC actively sense the lamina propria and luminal environment including the microbiota via receptors and transporters, subsequently mediating signals by secreting hormones and cytokines. Data indicate that immune cells and cytokine-mediated signaling impacts EEC numbers and function during infection and chronic inflammation of the gut, suggesting EEC not only to play a role in these pathologies but also being a target of inflammatory processes. This review presents data on the interrelation of incretins and inflammatory signaling. It focuses on the impact of intestinal inflammation, in particular inflammatory bowel disease, on EEC and the potential role of EEC and incretins in these pathologies. Furthermore, it highlights endoplasmic reticulum unfolded protein response, cytokines and the intestinal microbiota as possible targets of inflammatory and EEC signaling.

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1α and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.

The aim of the present study is to investigate the molecular pathways underlying amyotrophic lateral sclerosis (ALS) pathogenesis within the peripheral nervous system. We analyzed gene expression changes in human motor nerve diagnostic biopsies obtained from eight ALS patients and seven patients affected by motor neuropathy as controls. An integrated transcriptomics and system biology approach was employed. We identified alterations in the expression of 815 genes, with 529 up-regulated and 286 down-regulated in ALS patients. Up-regulated genes clustered around biological process involving RNA processing and protein metabolisms. We observed a significant enrichment of up-regulated small nucleolar RNA transcripts (p = 2.68*10-11) and genes related to endoplasmic reticulum unfolded protein response and chaperone activity. We found a significant down-regulation in ALS of genes related to the glutamate metabolism. Interestingly, a network analysis highlighted HDAC2, belonging to the histone deacetylase family, as the most interacting node. While so far gene expression studies in human ALS have been performed in postmortem tissues, here specimens were obtained from biopsy at an early phase of the disease, making these results new in the field of ALS research and therefore appealing for gene discovery studies.

Tumors successfully adapt to constantly changing intra- and extra-cellular environments, but the wirings of this process are still largely elusive. Here, we show that Heat Shock Protein 90 (HSP90)-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase (AMPK), inhibition of rapamycin-sensitive mTOR complex 1 (mTORC1), induction of autophagy, and expression of an endoplasmic reticulum (ER) unfolded protein response (UPR). This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial HSP90s are adaptive regulators of tumor bioenergetics, and tractable targets for cancer therapy.

CHOP (GADD153) is a small nuclear protein that dimerizes avidly with members of the C/EBP family of transcription factors. Normally undetectable, it is expressed at high levels in cells exposed to conditions that perturb protein folding in the endoplasmic reticulum and induce an endoplasmic reticulum stress response. CHOP expression in stressed cells is linked to the development of programmed cell death and, in some instances, cellular regeneration. In this study, representational difference analysis was used to compare the complement of genes expressed in stressed wild-type mouse embryonic fibroblasts with those expressed in cells nullizygous for chop. CHOP expression, in concert with a second signal, was found to be absolutely required for the activation by stress of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP). DOC4 is a mammalian ortholog of a Drosophila gene, Tenm/Odz, implicated in patterning of the early fly embryo, whereas DOC6 encodes a newly recognized homolog of the actin-binding proteins villin and gelsolin. These results reveal the existence of a novel CHOP-dependent signaling pathway, distinct from the known endoplasmic reticulum unfolded protein response, which may mediate changes in cell phenotype in response to stress.

Nevi and melanomas share the many of the same growth-promoting mutations. However, benign nevi eventually undergo growth arrest and stabilize while melanomas grow relentlessly. The difference in their long-term growth potential can in part be attributed to activation of cellular senescence pathways. The primary mediator of senescence in nevi appears to be p16. Redundant, secondary senescence systems are also present and include the p14-p53-p21 pathway, the IGFBP7 pathway, the FBXO31 pathway, and the PI3K-mediated stress-induced endoplasmic reticulum unfolded protein response. It is evident that these senescence pathways result in an irreversible arrest in most instances; however, they can clearly be overcome in melanoma. Circumvention of these pathways is most frequently associated with gene deletion or transcriptional repression. Reactivation of senescence mechanisms could serve to inhibit melanoma tumor progression.

Multiple Myeloma (MM) patients are vulnerable to infections, which remain a major cause of death, including early death. During infection, human immune cells sense the presence of invading pathogens through the toll-like receptor family (TLR) of receptors. TLRs detect microbes to activate transcriptional programs that orchestrate adaptive responses to specific insults. This means that they induce the endoplasmic reticulum (ER) unfolded protein response (UPR) to accommodate essential protein translation. If the UPR fails to resolve the protein-folding defect, due to severe and prolonged ER stress, apoptosis is activated. However, the timing sequence of the prolonged ER stress has probably implications for ER stress-induced apoptosis that might help cells to adapt under these conditions rather than driving them to apoptosis. Studies have shown that prolonged ER stress occurs in response to microbes and specifically when cells are exposed to lipopolysaccharide (LPS), a TLR4 activator. The prolonged stress, possibly arising from a massive increase in protein synthesis, has shown to suppress CHOP, an apoptosis biomarker, in ER-stressed macrophages, while low levels of CHOP expression promotes B cell survival. Expression and function of TLRs in MM has recently become the focus of several studies and although the regulatory role of TLRs in MM plasma cells has been reported, the underlying molecular mechanisms remain unclear. It has been shown that human myeloma cell lines (HMCL) and primary myeloma cells express high levels of TLRs and specifically of TLR-4 and TLR-9. The aim of our study was to investigate TLR4 signaling in myeloma cells and to explore possible implications with endoplasmic reticulum unfolded protein response as a potential mechanism of drug resistance. We initially investigated whether TLR-4 is expressed in human myeloma cell lines and primary myeloma cells and we found that TLR-4 mRNA is expressed at increased levels (2-10 fold) both in HMCLs and primary cells. To test the hypothesis that TLR-4 signaling may suppress CHOP expression during sustained UPR response, two myeloma cell lines, H929 and U266, were pre-treated with low dose LPS (1 ng/mL) and then subjected to ER stress conditions by treatment with tunicamycin (TM). LPS pre-treatment significantly decreased CHOP mRNA expression after 24 hours. Despite the marked suppression of CHOP, LPS pre-treatment of these myeloma cell lines did not suppress ATF4 mRNA levels which also were not altered by TM treatment. LPS pre-treatment did not also suppress XBP-1 splicing compared to the control ER-stressed cells. To test the specificity of these effects, the same set of experiments where performed on other cancer tissues such as ovarian cancer cell lines. Interestingly, although LPS pre-treatment increased TLR-4 mRNA expression in SKOV3 ovarian cancer cell line, CHOP mRNA levels remained intact prior and after treatment while TM treatment did not make any difference in CHOP mRNA expression. These results suggest the relevance of exploring this pathway in tissues such as plasma cells which are highly dependent on the UPR as a repair mechanism. Pre-treated LPS and TM samples of HMCLs were also subjected to Annexin-PI staining to determine the amount of apoptosis. As expected, pre-treated LPS myeloma cells which were exposed to TM had 30% lower Annexin-FITC stained cells compared to the TM-stressed cells only. These data suggest that blockage of CHOP by TLR4 ligands may promote the growth and survival of MM cells. We then examined the impact of therapy with bortezomib on TLR4 and CHOP mRNA expression in primary tumors cells which were collected before and at day 7 after bortezomib-based therapy from 6 myeloma patients. In 5 out of 6 cases TLR-4 expression was significantly up-regulated and was accompanied with a coupled down-regulation of CHOP mRNA expression. In conclusion, our data suggest that the TLR-4 signaling pathway might provide a translational control pathway which enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis. Further exploration of this pathway is needed to establish its role as a potential mechanism of drug resistance. Disclosures: No relevant conflicts of interest to declare.

Methods: MRNAs obtained from PBMCs were stimulated by LPS or left un-stimulated for 4h. We first used the Affymetrix Human Exon Array to analyze regulation of 14,851 and 15,334 expressed genes out of the 32,778 on the array in four patients and four ‘healthy’ subjects respectively. Results were validated using RTqPCR to measure expression of 63 genes of interest in a prospective cohort of 57 patients and 9 ‘healthy’ subjects. Results: Gene profiling revealed a core of 741 genes that were similarly regulated by LPS in cirrhotic and ‘healthy’ PBMCs. Among them, 300 LPS-induced genes were enriched in GO categories ‘regulation of mononuclear cell proliferation’ and ‘cytokine activity’. 441 shared LPS-downregulated genes were involved in recognition of PAMPs, chemotaxis, cell migration and lysosome suggesting that LPS-induced immune paralysis is a universal response. Comparison of functions associated with the 1,356 genes that were specifically regulated by LPS in cirrhotic cells to functions of the 1,049 genes specifically regulated in ‘healthy’ cells allowed to define a cirrhosis specific phenotype. Unlike healthy cells, LPS-stimulated cirrhotic cells did not exhibit the interferon (type I and II)-mediated program (e.g. IRF1, IRF2, IRF7, IFNG, IFI6, STAT1, STAT2, IFITM2), which is critical for immune host defense and resolution of inflammation. Consistent with these results, CXCL10 and CXCL11, two interferoninduced chemokines were induced by LPS in ‘healthy’ but not in cirrhotic PBMCs. Moreover, LPS-stimulated cirrhotic cells had downregulation of endocytic trafficking genes, which may results in decreased TLR4 endocytosis and inhibition of TRIF-mediated interferon pathway. Conclusions: Both LPS-stimulated cirrhotic and ‘healthy’ PBMCs exhibit features of immune paralysis. Moreover, LPS stimulated cirrhotic PBMCs have a specific defect of induction of interferon target genes involved in host defense and resolution of inflammation. These findings may explain the severity of bacterial infections in cirrhosis.