Cellular Stress Markers Research Articles

Love thy neighbor: Social buffering following exposure to an acute thermal stressor in a gregarious fish, the lake sturgeon (Acipenser fulvescens)

Social buffering is a phenomenon where the presence of conspecifics reduces an animal's stress response. Well known in mammals, social buffering was recently described in fishes exhibiting pronounced social hierarchies. Lake sturgeon (Acipenser fulvescens) are a gregarious rather than hierarchical fish. Therefore, we tested their capacity for social buffering following exposure to an acute thermal stress. Isolated or grouped (three or six similarly sized conspecifics) age-0 lake sturgeon were exposed to a critical thermal maximum (CTmax) test. We measured the endocrine and cellular response to acute thermal shock by assessing whole body cortisol concentration and mRNA expression of steroidogenic acute regulatory protein (StAR) and heat shock proteins (hsp90a, hsp90b, and hsp70) during recovery from the CTmax test. Isolation or grouping had no effect on CTmax. Whole body cortisol concentrations in isolated fish were approximately three-fold higher than in grouped fish 1 h post-CTmax and two-fold higher than grouped fish 20 h post-CTmax. Similarly, 1 h post-CTmax, mRNA expression of StAR, hsp90a, hsp90b and hsp70 were three to four-fold higher in isolated fish compared to groups of three and six fish. At 20 h post-CTmax, expression of StAR was approximately two-fold higher in isolated fish, but expression of hsp90a, hsp90b, and hsp70 was not significantly different between isolated and grouped fish. While conspecific presence had no effect on CTmax, the significant reduction of endocrine and cellular stress markers post-CTmax in grouped fish strongly suggests that lake sturgeon may use social buffering to combat potential deleterious effects of exposure to heat stress.

NP03, a Microdose Lithium Formulation, Blunts Early Amyloid Post-Plaque Neuropathology in McGill-R-Thy1-APP Alzheimer-Like Transgenic Rats.

Epidemiological, preclinical, and clinical studies have suggested a role for microdose lithium in reducing Alzheimer's disease (AD) risk by modulating key mechanisms associated with AD pathology. The novel microdose lithium formulation, NP03, has disease-modifying effects in the McGill-R-Thy1-APP transgenic rat model of AD-like amyloidosis at pre-plaque stages, before frank amyloid-β (Aβ) plaque deposition, during which Aβ is primarily intraneuronal. Here, we are interested in determining whether the positive effects of microdose lithium extend into early Aβ post-plaque stages. We administered NP03 (40μg Li/kg; 1 ml/kg body weight) to McGill-R-Thy1-APP transgenic rats for 12 weeks spanning the transition phase from plaque-free to plaque-bearing. The effect of NP03 on remote working memory was assessed using the novel object recognition task. Levels of human Aβ38, Aβ40, and Aβ42 as well as levels of pro-inflammatory mediators were measured in brain-extracts and plasma using electrochemiluminescent assays. Mature Aβ plaques were visualized with a thioflavin-S staining. Vesicular acetylcholine transporter (VAChT) bouton density and levels of chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-6 (IL-6), and 4-hydroxynonenal (4-HNE) were probed using quantitative immunohistochemistry. During the early Aβ post-plaque stage, we find that NP03 rescues functional deficits in object recognition, reduces loss of cholinergic boutons in the hippocampus, reduces levels of soluble and insoluble cortical Aβ42 and reduces hippocampal Aβ plaque number. In addition, NP03 reduces markers of neuroinflammation and cellular oxidative stress. Together these results indicate that microdose lithium NP03 is effective at later stages of amyloid pathology, after appearance of Aβ plaques.

Thymosin β4 cytoplasmic/nuclear translocation as a new marker of cellular stress. A Caco2 case study.

Biomarkers of cell stress are important for proper diagnosis, and in studies of how cells respond to drug treatment. Biomarkers that respond early to pharmacological treatment could improve therapy by tailoring the treatment to the needs of the patient. Thymosin beta-4 (Tβ4) plays a significant role in many aspects of cellular metabolism because of its actin-sequestering properties. Other physiological functions of Tβ4 have been also reported. Among these, Tβ4 may play a crucial role during cellular stress. We addressed the relevance of Tβ4 in cellular stress conditions by using different treatments (serum starvation, DMSO, and butyrate administration) in a colon adenocarcinoma cell line (CaCo2), a cell line frequently used for in vitro experimental studies of Tβ4. In this study, different stress stimuli were analyzed and the obtained results were compared using immunocytochemistry, and molecular and biochemical methods. Taken together, the data clearly indicate that the Tβ4 peptide is involved in adaptive and defensive cellular mechanisms, and that different stress inducers lead to a similar Tβ4 cytoplasmic/nuclear translocation. The translocation of Tβ4 between the cytoplasm and the nucleus of the cell seems characteristic of a possible molecular response to cellular stress exerted by this peptide.

Cold temperature tolerance of the alien Indo-Pacific damselfish Neopomacentrus cyanomos from the Southern Gulf of Mexico

The abundance of the alien, Indo-Pacific damselfish Neopomacentrus cyanomos on an oil-loading platform in the southwest Gulf of Mexico indicates that widely distributed platforms could facilitate the expansion of its geographic range across the western and northern fringes of the Gulf. From there it likely will spread to other areas of the Greater Caribbean. The lionfish example demonstrates that it eventually happens, and can do so rapidly. Reduced temperature effects on the physiology of this species were examined to better predict its survivability in the northern Gulf during winter, when sea surface temperatures fall as low as 15 °C along the coast. Overall, our results show that when the degree of experimental temperature decline was large and rapid, no compensation occurred and the stress response observed mostly reflected cellular processes that minimized damage. Integrated biomarker response values were significantly different between fish rapidly exposed to colder vs. warmer temperatures (declines of −4 °C each day, from 26 to 14 °C), reflected in higher values of blood metabolites and routine metabolic rates observed in fish exposed to 14 and 18 °C respectively, and lower activity of all enzymes, lower protein carbonylation, and higher oxidative damage to lipids in fish exposed to 14 °C. While the physiological proxies responded to minimize damage during the rapid-decrease experiment, the same proxies reflected the consequences of compensation when fish were thermally challenged after a 45 days acclimation at 18 °C. In this case, lower values of blood metabolites and high antioxidant levels and indicators of damages underpinned its pejus lower range. Based on the results of the present work, it seems clear that low winter SSTs in the northern Gulf will slow down the colonization of the inshore area of N. cyanomos. We suggest that the use of physiological cellular stress markers on specimens acquired at the beginning of an invasion should be implemented in new standardized experimental protocols, including both rapid increases/decreases of temperature and post-acclimation temperature challenges, to assess the invasiveness potential of aquatic species such as this.

Cell Stress Reduction by a Novel Perfusion-Culture System Using Commercial Culture Dish

Two typical medium replacement methods are employed to culture cells, that is, traditional manual medium change, wherein complete medium changes are performed at short-term intervals; and perfusion, wherein continuous culture media delivery and discharge are performed. The former is associated with the issue of readily accumulation of metabolic waste products, which is resolved by the latter. However, the latter requires a specific chamber for cell culturing such as a microfluidic channel for flow stability. Generally, bio-medical researchers require a culturing process that utilizes commercial culture dishes to test their conventional manipulation know-how, experience, and protocols. Thus, we constructed a perfusion-culture system using commercial 35 mm culture dishes. To use commercial culture dishes, it is necessary to maintain the culturing conditions including a constant volume and uniform flow rate, and we invented a novel adapter, culture dish adapter (CD-Adapter), attached to a commercial culture dish. The CD-Adapter is fixed with a holding jig to keep the medium volume constant, so it can maintain a uniform flow in the culture dish. Additionally, we demonstrate the applicability of the system by applying it to a perfusion culture of 293T cells, revealing a 50% reduction in DDIT3, a cellular stress marker, compared with that in conventional manual medium change.

Screening of Cellular Stress Responses Induced by Ambient Aerosol Ultrafine Particle Fraction PM0.5 in A549 Cells.

Effects of airborne particles on the expression status of markers of cellular toxic stress and on the release of eicosanoids, linked with inflammation and oxidative damage, remain poorly characterized. Therefore, we proposed a set of various methodological approaches in order to address complexity of PM0.5-induced toxicity. For this purpose, we used a well-characterized model of A549 pulmonary epithelial cells exposed to a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Electron microscopy confirmed accumulation of PM0.5 within A549 cells, yet, autophagy was not induced. Expression profiles of various cellular stress response genes that have been previously shown to be involved in early stress responses, namely unfolded protein response, DNA damage response, and in aryl hydrocarbon receptor (AhR) and p53 signaling, were analyzed. This analysis revealed induction of GREM1, EGR1, CYP1A1, CDK1A, PUMA, NOXA and GDF15 and suppression of SOX9 in response to PM0.5 exposure. Analysis of eicosanoids showed no oxidative damage and only a weak anti-inflammatory response. In conclusion, this study helps to identify novel gene markers, GREM1, EGR1, GDF15 and SOX9, that may represent a valuable tool for routine testing of PM0.5-induced in vitro toxicity in lung epithelial cells.

Circulating levels of GDF15 in patients with myalgic encephalomyelitis/chronic fatigue syndrome

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterised by fatigue and post-exertional malaise. Its pathogenesis is poorly understood. GDF15 is a circulating protein secreted by cells in response to a variety of stressors. The receptor for GDF15 is expressed in the brain, where its activation results in a range of responses. Among the conditions in which circulating GDF15 levels are highly elevated are mitochondrial disorders, where early skeletal muscle fatigue is a key symptom. We hypothesised that GDF15 may represent a marker of cellular stress in ME/CFS.MethodsGDF15 was measured in serum from patients with ME/CFS (n = 150; 100 with mild/moderate and 50 with severe symptoms), “healthy volunteers” (n = 150) and a cohort of patients with multiple sclerosis (n = 50).ResultsCirculating GDF15 remained stable in a subset of ME/CFS patients when sampled on two occasions ~ 7 months (IQR 6.7–8.8) apart, 720 pg/ml (95% CI 625–816) vs 670 pg/ml (95% CI 598–796), P = 0.5. GDF15 levels were 491 pg/ml in controls (95% CI 429–553), 546 pg/ml (95% CI 478–614) in MS patients, 560 pg/ml (95% CI 502–617) in mild/moderate ME/CFS patients and 602 pg/ml (95% CI 531–674) in severely affected ME/CFS patients. Accounting for potential confounders, severely affected ME/CFS patients had GDF15 concentrations that were significantly increased compared to healthy controls (P = 0.01). GDF15 levels were positively correlated (P = 0.026) with fatigue scores in ME/CFS.ConclusionsSevere ME/CFS is associated with increased levels of GDF15, a circulating biomarker of cellular stress that appears which stable over several months.

Abstract A110: Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells

Abstract Androgen receptor (AR) over-expression, mutation and the emergence of AR splice variants (AR-Vs) promote disease-driving AR signaling in castration-resistant prostate cancer (CRPC). In addition to the sustained AR activation, apoptosis evasion adds extra hurdle for treating hormone refractory prostate cancer (PCa). To overcome these challenges, we firstly designed isothiocyanate (ITC)-containing hybrid AR antagonist (ITC-ARi) and then rationally combined ITC-ARi with glutathione (GSH) biosynthesis inhibitor buthionine sulphoximine (BSO) to efficiently downregulate AR/AR-V and induce ferroptosis in enzalutamide (Enz)-resistant PCa cells. Naturally occurring ITCs (e.g., sulforaphane and phenylethyl isothiocyanate) display pleotropic anti-PCa activities. A representative ITC-ARi 2-63 is designed by incorporating ITC into an AR ligand scaffold. The sulfhydryl reactivity of ITC is transiently masked as N-acetyl cysteine (NAC) conjugate that gradually releases parental free ITC in aqueous solution. 2-63 antagonizes AR transactivation, downregulates AR/AR-V7, and upregulates cellular stress markers Hsp70 and heme oxygenase-1 (HO-1), indicating the disruption of heat shock proteins (Hsps) and the activation of Nrf2 pathway. Because the anticancer effects of ITC/ITC-NAC conjugate are attenuated by GSH-participated conjugation or thiol exchange, we rationally combined 2-63 with BSO for enhanced anti-PCa activities. 2-63 and BSO synergistically reduce viability of multiple PCa cell lines (VCaP, C4-2 and 22Rv-1), and the noncancerous prostatic RWPE-1 cell is much less affected. Because 2-63 plus BSO does not upregulate typical apoptosis markers, we investigated alternative cell death mechanism in combinatorial treatment. Drug combination-caused cell viability loss is effectively rescued by iron chelator deferoxamine (DFO), antioxidants α-tocopherol (α-Toc) and ferrostatin-1 (Fer-1), or the inhibitor of HO-1 Zinc (II) protoporphyrin IX (ZnPP). Both Fer-1 and α-Toc are potent radical scavengers blocking lipid hydroperoxide generation, and ZnPP suppresses HO-1-catalyzed upregulation of intracellular Fe2+. The reverse of growth suppression by DFO, antioxidants and ZnPP supports the induction of ferroptosis, a regulated non-apoptotic cell death caused by reactive oxygen species (ROS)- and iron-dependent lipid peroxidation. 2-63 and BSO also more efficiently downregulate AR/AR-V than 2-63 alone in Enz-resistant PCa cells. Interestingly, ferroptosis-preventing DFO and Fer-1 do not rescue AR/AR-V7 and are unable to suppress the upregulation of Hsp70 and HO-1. Our results suggest that the synergism of 2-63 and BSO occurs through increasing drug accessibility to cellular targets, expanding availability of iron and potentially affecting glutathione peroxidase 4 (GPX4) activity, the most important cellular defense to eliminate lipid hydroperoxide. Combining ITC-ARi and GSH-depleting agents could be a new concept leading to effective CRPC treatment. Citation Format: Zhihui Qin, Liping Xu, Siyu Ou. Isothiocyanate-containing hybrid androgen receptor (AR) antagonist downregulates AR and induces ferroptosis in GSH–deficient prostate cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A110. doi:10.1158/1535-7163.TARG-19-A110

A proteome-wide assessment of the oxidative stress paradigm for metal and metal-oxide nanomaterials in human macrophages

Responsible implementation of engineered nanomaterials (ENMs) into commercial applications is an important societal issue, driving demand for new approaches for rapid and comprehensive evaluation of their bioactivity and safety. An essential part of any research focused on identifying potential hazards of ENMs is the appropriate selection of biological endpoints to evaluate. Herein, we use a tiered strategy employing both targeted biological assays and untargeted quantitative proteomics to elucidate the biological responses of human THP-1 derived macrophages across a library of metal/metal oxide ENMs, raised as priority ENMs for investigation by NIEHS's Nanomaterial Health Implications Research (NHIR) program. Our results show that quantitative cellular proteome profiles readily distinguish ENM types based on their cytotoxic potential according to induction of biological processes and pathways involved in the cellular antioxidant response, TCA cycle, oxidative stress, endoplasmic reticulum stress, and immune responses as major processes impacted. Interestingly, bioinformatics analysis of differentially expressed proteins also revealed new biological processes that were influenced by all ENMs independent of their cytotoxic potential. These included biological processes that were previously implicated as mechanisms cells employ as adaptive responses to low levels of oxidative stress, including cell adhesion, protein translation and protein targeting. Unsupervised clustering revealed the most striking proteome changes that differentiated ENM classes highlight a small subset of proteins involved in the oxidative stress response (HMOX1), protein chaperone functions (HS71B, DNJB1), and autophagy (SQSTM), providing a potential new panel of markers of ENM-induced cellular stress. To our knowledge, the results represent the most comprehensive profiling of the biological responses to a library of ENMs conducted using quantitative mass spectrometry-based proteomics. The results provide a basis to identify the patterns of a diverse set of cellular pathways and biological processes impacted by ENM exposure in an important immune cell type, laying the foundation for multivariate, pathway-level structure activity assessments of ENMs in the future.

Flavonoids reduces lipopolysaccharide-induced release of inflammatory mediators in human bronchial epithelial cells: Structure-activity relationship

Flavonoids are polyphenolic compounds that are widely present in food and Chinese medicine. The aim of the present study was to identify the flavonoids with anti-inflammatory effects in the airway; and to determine the role of anti-oxidant and cyclic adenosine monophosphate (cAMP) in the anti-inflammatory effect. Human bronchial epithelial BEAS-2B cells were exposed to bacterial endotoxin lipopolysaccharide (LPS) in the absence or presence of different flavonoids, which are categorized according to their chemical structures in seven subclasses [anthocyanidins, chalcones, flavanes, flavanones, flavones, flavonols, isoflavones]. Among the 17 flavonoids tested, only apigenin (flavones), luteolin (flavones), daidzein (isoflavones) and genistein (isoflavones) reduced LPS-induced release of inflammatory cytokines/chemokines interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 in BEAS-2B cells. Quercetin caused further increase in LPS-induced IL-6 and IL-8 levels. It alone significantly increased nuclear factor-kappa B (NF-κB) p65 activity and the cellular oxidative stress marker malondialdehyde (MDA) level in BEAS-2B cells. By contrast, apigenin and genistein reduced LPS-induced increases in nuclear NF-κB activity and MDA level. Apigenin and genistein, but not quercetin, increased the cAMP level in BEAS-2B cells, and the cell-permeable cAMP analogue, 8-Br-cAMP, inhibited LPS-induced increase of IL-8 level. These findings suggest that the presence of C5–OH, C7–OH, C2=C3 and C4=O functional groups in the flavonoids is associated with greater anti-inflammatory effect, while that of C3–OH or glycosylation group at the A-ring greatly decreased the anti-inflammatory effect. The anti-inflammatory effect of these flavonoids may be related to their anti-oxidant properties, and partly to their ability in increasing cAMP level.

Adverse Childhood Experiences: Implications for Offspring Telomere Length and Psychopathology.

Adverse childhood experiences (ACEs) are associated with mental and physical health risks that, through biological and psychosocial pathways, likely span generations. Within an individual, telomere length (TL), an established marker of cellular stress and aging, is associated with both ACE exposure and psychopathology, providing the basis for an emerging literature suggesting that TL is a biomarker of the health risks linked to early-life adversity both within and across generations. The authors tested the effect of maternal ACEs on both the trajectory of infant TL and infant social-emotional problems at 18 months of age. Pregnant women were recruited, and maternal scores on the Adverse Childhood Experience questionnaire were obtained, along with demographic and prenatal stress measures. Postnatal visits with 155 mother-infant dyads occurred when infants were 4, 12, and 18 months of age. At each visit, infant buccal swabs were collected for TL measurement, and mothers completed measures of maternal depression. Mothers also completed the Child Behavior Checklist at the 18-month visit. Mixed-effects modeling was used to test how maternal ACEs influenced infant TL trajectory. Linear regression was used to test the association between maternal ACEs and infant internalizing and externalizing behaviors. Finally, the interaction between telomere attrition from 4 to 18 months and maternal ACEs was examined as a predictor of infant scores on the Child Behavior Checklist. Higher maternal ACEs were associated with shorter infant TL across infancy and higher infant externalizing behavioral problems at 18 months. No associations were found with internalizing behavioral problems. Telomere attrition from 4 to 18 months interacted with maternal ACEs to predict externalizing behaviors. In infants whose mothers reported higher scores on the Adverse Childhood Experience questionnaire, greater telomere attrition predicted higher externalizing problems, even when accounting for maternal postnatal depression and prenatal stress. These data demonstrate an interactive pathway between maternal early-life adversity and infant TL that predicts emerging behavioral problems in the next generations.

Patients with Incompetent Valves in Chronic Venous Insufficiency Show Increased Systematic Lipid Peroxidation and Cellular Oxidative Stress Markers.

Chronic venous insufficiency (CVI) is a disease that impacts cellular homeostasis. CVI may occur with a valvular destruction process known as venous reflux or valvular incompetence. One of the cellular processes that may be triggered as a consequence of these events is the production of reactive oxygen species (ROS), which may trigger the production of different cellular markers and cell damage processes, such as lipid peroxidation. Therefore, the present study performed an observational, analytical, and prospective cohort study by reviewing 110 patients with CVI, and the activities and plasma levels of iNOS, eNOS, NOX1, and NOX2 were determined using immunohistochemistry and RT-qPCR. Lipid peroxidation (MDA) was also measured. Patients were distributed according to the presence or absence of valvular incompetence-venous reflux, which was diagnosed clinically as the absence of venous reflux (NR = 29) or presence of venous reflux (R = 81). Each group was divided according to age, with a cutoff point of fifty years (NR < 50 = 13, NR ≥ 50 = 16, R < 50 = 32, and R ≥ 50 = 49). The results showed that R patients exhibited significantly increased plasma MDA levels, and R < 50 patients exhibited the highest statistically significant increase. iNOS, NOX1, and NOX2 exhibited the highest gene and protein expression in R patients. The increased expression was maintained in the R < 50 patients. Our data suggest that young patients with valvular incompetence (venous reflux) show higher levels of lipid peroxidation and oxidative stress, which reflects the characteristics of an aged patient.

A Novel Role for Somatostatin in the Survival of Mouse Pancreatic Beta Cells.

Cross-talk between different pancreatic islet cell types regulates islet function and somatostatin (SST) released from pancreatic delta cells inhibits insulin secretion from pancreatic beta cells. In other tissues SST exhibits both protective and pro-apoptotic properties in a tissue-specific manner, but little is known about the impact of the peptide on beta cell survival. Here we investigate the specific role of SST in the regulation of beta cell survival in response to physiologically relevant inducers of cellular stress including palmitate, cytokines and glucose. Pancreatic MIN6 beta cells and primary mouse islet cells were pre-treated with SST with or without the Gi/o signalling inhibitor, pertussis toxin, and exposed to different cellular stress factors. Apoptosis and proliferation were assessed by measurement of caspase 3/7 activity, TUNEL and BrdU incorporation, respectively, and expression of target genes was measured by qPCR. SST partly alleviated upregulation of cellular stress markers (Hspa1a and Ddit3) and beta cell apoptosis in response to factors such as lipotoxicity (palmitate), pro-inflammatory cytokines (IL1β and TNFα) and low glucose levels. This effect was mediated via a Gi/o protein-dependent pathway, but did not modify transcriptional upregulation of the specific NFκB-dependent genes, Nos2 and Ccl2, nor was it associated with transcriptional changes in SST receptor expression. Our results suggest an underlying protective effect of SST which modulates the beta cell response to ER stress and apoptosis induced by a range of cellular stressors associated with type 2 diabetes.

The Adaptive Proline Response in P. falciparum Is Independent of PfeIK1 and eIF2α Signaling.

We have previously identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum as the functional target of the natural product febrifugine and its synthetic analogue halofuginone (HFG), one of the most potent antimalarials discovered to date. However, our studies also discovered that short-term treatment of asexual blood stage P. falciparum with HFG analogues causes a 20-fold increase in intracellular proline, termed the adaptive proline response (APR), which renders parasites tolerant to HFG. This novel resistance phenotype lacks an apparent genetic basis but remains stable after drug withdrawal. On the basis of our findings that HFG treatment induces eIF2α phosphorylation, a sensitive marker and mediator of cellular stress, we here investigate if eIF2α-signaling is functionally linked to the APR. In our comparative studies using a parasite line lacking PfeIK1, the Plasmodium orthologue of the eIF2α-kinase GCN2 that mediates amino acid deprivation sensing, we show that HFG activity and the APR are independent from PfeIK1 and eIF2α signaling.

The anti-proliferative effects of adiponectin on human lung adenocarcinoma A549 cells and oxidative stress involvement

Adiponectin (Acrp30) plays an important role in energy metabolism and inflammation. Recently, in vivo serum Acrp30 levels have been reported to be correlated to risk of developing several types of cancers such as lung cancer, and in vitro studies have demonstrated a role for Acrp30 in the control of cell proliferation and survival. However, the molecular effects of Acrp30 on lung cancer have not yet been clearly defined.In the present study, we investigated the effects of different concentrations of Acrp30 on the A549 human alveolar epithelial cell line, an in vitro model of lung adenocarcinoma. A549 cells were exposed to various concentrations of Acrp30 and successively, proliferation, apoptosis and oxidative stress were evaluated by MTT test, caspase activity assay, flow-cytometry and western blotting analysis. Our results demonstrated that Acrp30 causes, in a time- and dose-dependent manner, a reduction of cell viability and duplication together with an increase in cell apoptosis rate. In addition, we found that Acrp30 induces an increase of lipid peroxidation evaluated by TBARS assay and a concomitant reduction of nitric oxide release, both markers of cellular oxidative stress. Taken together, our data on A549 cells provides new insight into potential involvement of Acrp30 on physio-pathologic mechanisms of lung diseases through interference with proliferation, apoptosis and oxidative status.

Evaluation of the role of interesterified fat in metabolic parameters and cellular adipose tissue stress pathways

The western lifestyle is characterized with increased intake of processed and ultraprocessed foods, which have trans fat in their composition. As an alternative to the of trans fat, interesterification is one of the process that have being used by the food industry for the production of plastic fats and trans free. Thus, the aim of this study was to evaluate the role of interesterified fat on metabolic parameters, lipid profile and cellular stress markers in white adipose tissue in mice.

Differential regulation of cellular stress responses by the endoplasmic reticulum-resident Selenoprotein S (Seps1) in proliferating myoblasts versus myotubes.

The antioxidant Selenoprotein S (Seps1, Selenos) is an endoplasmic reticulum (ER)‐resident protein associated with metabolic and inflammatory disease. While Seps1 is highly expressed in skeletal muscle, its mechanistic role as an antioxidant in skeletal muscle cells is not well characterized. In C2C12 myotubes treated with palmitate for 24 h, endogenous Seps1 protein expression was upregulated twofold. Two different siRNA constructs were used to investigate whether decreased levels of Seps1 exacerbated lipid‐induced oxidative and ER stress in C2C12 myotubes and myoblasts, which differ with regards to cell cycle state and metabolic phenotype. In myoblasts, Seps1 protein knockdown of ~50% or ~75% exacerbated cellular stress responses in the presence of palmitate; as indicated by decreased cell viability and proliferation, higher H2O2 levels, a lower reduced to oxidized glutathione (GSH:GSSG) ratio, and enhanced gene expression of ER and oxidative stress markers. Even in the absence of palmitate, Seps1 knockdown increased oxidative stress in myoblasts. Whereas, in myotubes in the presence of palmitate, a ~50% knockdown of Seps1 was associated with a trend toward a marginal (3‐5%) decrease in viability (P = 0.05), decreased cellular ROS levels, and a reduced mRNA transcript abundance of the cellular stress marker thioredoxin inhibitory binding protein (Txnip). Furthermore, no enhancement of gene markers of ER stress was observed in palmitate‐treated myotubes in response to Seps1 knockdown. In conclusion, reduced Seps1 levels exacerbate nutrient‐induced cellular stress responses to a greater extent in glycolytic, proliferating myoblasts than in oxidative, differentiated myotubes, thus demonstrating the importance of cell phenotype to Seps1 function.

Increased tolerance to organic xenobiotics following recent allopolyploidy in Spartina (Poaceae)

Genome doubling or polyploidy is a widespread phenomenon in plants where it has important evolutionary consequences affecting the species distribution and ecology. PAHs are ubiquitous organic pollutants, which represent a major environmental concern. Recent data showed that tolerance to organic xenobiotics involve specific signaling pathways, and detoxifying gene sets referred as ‘the xenome’. However, no data are available about how polyploidy impacts tolerance to organic xenobiotics. In the present paper, we investigated PAH tolerance following allopolyploidization in Spartina alterniflora, S. maritima and their derived allopolyploid species S. anglica. We performed comparative analyses of cellular compartmentalization, photosynthetic indices, and oxidative stress markers under phenanthrene-induced stress, and found that S. anglica exhibit increased tolerance compared to its parents. Based on 52 genes potentially involved in phenanthrene detoxification previously identified in A. thaliana, we investigated the Spartina xenome using genomic and transcriptomic available resources. Subsequently, we focused on GSTs, a ubiquitous enzymes class involved in organic xenobiotic detoxification. We examined expression profiles of selected genes by RT-qPCR, and revealed various patterns of parental expression alteration in the allopolyploid. The impacts of allopolyploidization on phenanthrene-induced stress and their potential ecological implications are discussed. The neo-allopolyploid S. anglica appears as a potential candidate for phytoremediation in PAH-polluted marshes.

The Spectrum of Neurological and White Matter Changes and Premutation Status Categories of Older Male Carriers of the FMR1 Alleles Are Linked to Genetic (CGG and FMR1 mRNA) and Cellular Stress (AMPK) Markers.

The fragile X premutation (PM) allele contains a CGG expansion of 55–200 repeats in the FMR1 gene’s promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39–81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers – CGG repeat number and the levels of the FMR1 mRNA – were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85–90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings – suggestive of a role of this enzyme in the risk of FXTAS – need to be verified and expanded in future studies using larger samples and longitudinal assessment.

Abstract 290: Fibroblast Growth Factor 21 After Out-of-Hospital Cardiac Arrest

Introduction: Fibroblast growth factor (FGF) 21 is a novel marker of mitochondrial and cellular stress. Due to the pivotal role of mitochondria in oxidative metabolism and oxidative stress we tested the hypothesis that FGF 21 would reflect the severity of hypoxia-reperfusion injury after cardiac arrest. Methods: We measured serum concentration of FGF 21 upon ICU admission and 24, 48 and 72 hours after out-of hospital cardiac arrest (OHCA) in 112 patients. All patients had ventricular fibrillation/tachycardia (VF/VT) as initial rhythm and received targeted temperature management (TTM) of 33 or 36 °C for 24 hours. We defined 6-month Cerebral Performance Category (CPC) 1-2 as good and 3-5 as poor neurologic outcome. We used samples from 40 age- and gender-matched non-critically ill emergency room patients as controls. Group differences were assessed with the Mann Whitney U test and temporal differences with Friedman’s ANOVA and Wilcoxon signed ranks test. Results: The median (inter-quartile range, IQR) FGF 21 concentration at ICU admission was 790 (370-1600) pg/ml and the peak value was 1200 (460-2500) pg/ml at 48 hours. Compared to the ICU admission values, the concentrations in control samples [250 (94-910) pg/ml] were significantly lower (P &lt; 0.001). The ICU admission FGF 21 levels were higher in patients with poor neurologic outcome (P = 0.04) and they correlated weakly with the time from collapse to return of spontaneous circulation (Spearman’s rho 0.23). Serum FGF 21 levels changed over time (P &lt; 0.001). Figure 1 presents serum FGF 21 median levels with IQR (box) and 10-90 percentiles (whiskers). Conclusion: Patients after OHCA have elevated FGF 21 levels after successful resuscitation. Higher ICU admission FGF 21 levels associate with poor 6-month neurologic outcome.