Showing papers in "Cell Stress & Chaperones in 2018"

Melatonin protected cardiac microvascular endothelial cells against oxidative stress injury via suppression of IP3R-[Ca 2+ ]c/VDAC-[Ca 2+ ]m axis by activation of MAPK/ERK signaling pathway

Abstract: The cardiac microvascular reperfusion injury is characterized by the microvascular endothelial cells (CMECs) oxidative damage which is responsible for the progression of cardiac dysfunction. However, few strategies are available to reverse such pathologies. This study aimed to explore the mechanism by which oxidative stress induced CMECs death and the beneficial actions of melatonin on CMECs survival, with a special focused on IP3R-[Ca2+]c/VDAC-[Ca2+]m damage axis and the MAPK/ERK survival signaling. We found that oxidative stress induced by H2O2 significantly activated cAMP response element binding protein (CREB) that enhanced IP3R and VDAC transcription and expression, leading to [Ca2+]c and [Ca2+]m overload. High concentration of [Ca2+]m suppressed ΔΨm, opened mPTP, and released cyt-c into cytoplasm where it activated mitochondria-dependent death pathway. However, melatonin could protect CMECs against oxidative stress injury via stimulation of MAPK/ERK that inactivated CREB and therefore blocked IP3R/VDAC upregulation and [Ca2+]c/[Ca2+]m overload, sustaining mitochondrial structural and function integrity and ultimately blockading mitochondrial-mediated cellular death. In summary, these findings confirmed the mechanisms by which oxidative injury induced CMECs mitochondrial-involved death and provided an attractive and effective way to enhance CMECs survival.

Heat shock proteins and DNA repair mechanisms: an updated overview

Abstract: Heat shock proteins (HSPs), also known as molecular chaperones, participate in important cellular processes, such as protein aggregation, disaggregation, folding, and unfolding. HSPs have cytoprotective functions that are commonly explained by their antiapoptotic role. Their involvement in anticancer drug resistance has been the focus of intense research efforts, and the relationship between HSP induction and DNA repair mechanisms has been in the spotlight during the past decades. Because DNA is permanently subject to damage, many DNA repair pathways are involved in the recognition and removal of a diverse array of DNA lesions. Hence, DNA repair mechanisms are key to maintain genome stability. In addition, the interactome network of HSPs with DNA repair proteins has become an exciting research field and so their use as emerging targets for cancer therapy. This article provides a historical overview of the participation of HSPs in DNA repair mechanisms as part of their molecular chaperone capabilities.

Methods to validate Hsp90 inhibitor specificity, to identify off-target effects, and to rethink approaches for further clinical development.

Abstract: The molecular chaperone Hsp90 is one component of a highly complex and interactive cellular proteostasis network (PN) that participates in protein folding, directs misfolded and damaged proteins for destruction, and participates in regulating cellular transcriptional responses to environmental stress, thus promoting cell and organismal survival. Over the last 20 years, it has become clear that various disease states, including cancer, neurodegeneration, metabolic disorders, and infection by diverse microbes, impact the PN. Among PN components, Hsp90 was among the first to be pharmacologically targeted with small molecules. While the number of Hsp90 inhibitors described in the literature has dramatically increased since the first such small molecule was described in 1994, it has become increasingly apparent that not all of these agents have been sufficiently validated for specificity, mechanism of action, and lack of off-target effects. Given the less than expected activity of Hsp90 inhibitors in cancer-related human clinical trials, a re-evaluation of potentially confounding off-target effects, as well as confidence in target specificity and mechanism of action, is warranted. In this commentary, we provide feasible approaches to achieve these goals and we discuss additional considerations to improve the clinical efficacy of Hsp90 inhibitors in treating cancer and other diseases.

Extracellular small heat shock proteins: exosomal biogenesis and function.

Abstract: Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.

The small heat shock protein B8 (HSPB8) efficiently removes aggregating species of dipeptides produced in C9ORF72-related neurodegenerative diseases

Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not “classical” misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.

Amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD) are characterised by differential activation of ER stress pathways: focus on UPR target genes

Abstract: The endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR), which is strongly activated in most neurodegenerative disorders. UPR signalling pathways mediated by IRE1α and ATF6 play a crucial role in the maintenance of ER homeostasis through the transactivation of an array of transcription factors. When activated, these transcription factors induce the expression of genes involved in protein folding and degradation with pro-survival effects. However, the specific contribution of these transcription factors to different neurodegenerative diseases remains poorly defined. Here, we characterised 44 target genes strongly influenced by XBP1 and ATF6 and quantified the expression of a subset of genes in the human post-mortem spinal cord from amyotrophic lateral sclerosis (ALS) cases and in the frontal and temporal cortex from frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD) cases and controls. We found that IRE1α-XBP1 and ATF6 pathways were strongly activated both in ALS and AD. In ALS, XBP1 and ATF6 activation was confirmed by a substantial increase in the expression of both known and novel target genes involved particularly in co-chaperone activity and ER-associated degradation (ERAD) such as DNAJB9, SEL1L and OS9. In AD cases, a distinct pattern emerged, where targets involved in protein folding were more prominent, such as CANX, PDIA3 and PDIA6. These results reveal that both overlapping and disease-specific patterns of IRE1α-XBP1 and ATF6 target genes are activated in AD and ALS, which may be relevant to the development of new therapeutic strategies. Graphical abstract The endoplasmic reticulum (ER) plays an important role in maintenance of proteostasis through the unfolded protein response (UPR). Two major UPR signalling pathways are mediated by IRE1α and ATF6. Here, we demonstrate that these pathways activate differential gene sets in human post-mortem tissues derived from amyotrophic lateral sclerosis (ALS) compared to Alzheimer's disease (AD) cases. Our results identify IRE1α and ATF6 specific targets that can have major implications in the development of new therapeutic strategies and potential biomarkers.

The role of αB-crystallin in skeletal and cardiac muscle tissues.

Abstract: All organisms and cells respond to various stress conditions such as environmental, metabolic, or pathophysiological stress by generally upregulating, among others, the expression and/or activation of a group of proteins called heat shock proteins (HSPs). Among the HSPs, special attention has been devoted to the mutations affecting the function of the αB-crystallin (HSPB5), a small heat shock protein (sHsp) playing a critical role in the modulation of several cellular processes related to survival and stress recovery, such as protein degradation, cytoskeletal stabilization, and apoptosis. Because of the emerging role in general health and disease conditions, the main objective of this mini-review is to provide a brief account on the role of HSPB5 in mammalian muscle physiopathology. Here, we report the current known state of the regulation and localization of HSPB5 in skeletal and cardiac tissue, making also a critical summary of all human HSPB5 mutations known to be strictly associated to specific skeletal and cardiac diseases, such as desmin-related myopathies (DRM), dilated (DCM) and restrictive (RCM) cardiomyopathy. Finally, pointing to putative strategies for HSPB5-based therapy to prevent or counteract these forms of human muscular disorders.

Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus.

Abstract: Bovine milk is rich in exosomes, which contain abundant miRNAs and play important roles in the regulation of neonatal growth and development of adaptive immunity. Here, we analyzed miRNA expression profiles of bovine milk exosomes from three healthy and three mastitic cows, and then six miRNA libraries were constructed. Interestingly, we detected no scRNAs and few snRNAs in milk exosomes; this result indicated a potential preference for RNA packaging in milk exosomes. A total of 492 known and 980 novel exosomal miRNAs were detected, and the 10 most expressed miRNAs in the six samples accounted for 80-90% of total miRNA-associated reads. Expression analyses identified 18 miRNAs with significantly different expression between healthy and infected animals; the predicted target genes of differentially expressed miRNAs were significantly enriched in immune system process, response to stimulus, growth, etc. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including inflammatory, immune, and cancer pathways. Our survey provided comprehensive information about milk exosomes and exosomal miRNAs involved in mastitis. Moreover, the differentially expressed miRNAs, especially miR-223 and miR-142-5p, could be considered as potential candidates for mastitis.

Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways.

Abstract: Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells

Abstract: Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy.

Abstract: Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis However, little is known about the molecular mechanisms of PA toxicity In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process

Melatonin alleviates inflammation-induced apoptosis in human umbilical vein endothelial cells via suppression of Ca 2+ -XO-ROS-Drp1-mitochondrial fission axis by activation of AMPK/SERCA2a pathway

Abstract: Endothelia inflammation damage is vital to the development and progression of chronic venous disease. In the present study, we explored the protective effect of melatonin on endothelia apoptosis induced by LPS, particularly focusing on the mitochondrial fission. We demonstrated that human umbilical vein endothelial cells (HUVEC) subjected to LPS for 12 h exhibited a higher apoptotic rate. However, melatonin (1–20 μM) treatment 12 h before LPS had the ability to protect HUVEC cell against LPS-mediated apoptosis in a dose-dependent manner. Furthermore, LPS induced the cytoplasmic calcium overload which was responsible for the upregulation of calcium-dependent xanthine oxidase (XO). Higher XO expression was associated with reactive oxygen species (ROS) overproduction, leading to the Drp1 phosphorylation at the Ser616 site and migration on the surface of mitochondria. Furthermore, phosphorylated Drp1 initiated the mitochondrial fission contributing to the caspase9-dependent mitochondrial apoptosis as evidenced by lower membrane potential, more cyt-c leakage into the nuclear, and higher expression of proapoptotic proteins. However, melatonin treatment could trigger the AMPK pathway, which was followed by the increased SERCA2a expression. Activation of AMPK/SERCA2a by melatonin inhibited the calcium overload, XO-mediated ROS outburst, Drp1-required mitochondrial fission, and final mitochondrial apoptosis. In summary, this study confirmed that LPS induced HUVEC apoptosis through Ca2+-XO-ROS-Drp1-mitochondrial fission axis and that melatonin reduced the apoptosis of HUVEC through activation of the AMPK/SERCA2a pathway.

Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken

Abstract: Thermal manipulation during embryogenesis has been demonstrated to enhance the thermotolerance capacity of broilers through epigenetic modifications. Heat shock proteins (HSPs) are induced in response to stress for guarding cells against damage. The present study investigates the effect of thermal conditioning during embryogenesis and thermal challenge at 42 days of age on HSP gene and protein expression, DNA methylation and in vitro luciferase assay in brain tissue of Naked Neck (NN) and Punjab Broiler-2 (PB-2) chicken. On the 15th day of incubation, fertile eggs from two breeds, NN and PB-2, were randomly divided in to two groups: control (C)-eggs were incubated under standard incubation conditions, and thermal conditioning (TC)-eggs were exposed to higher incubation temperature (40.5°C) for 3 h on the 15th, 16th, and 17th days of incubation. The chicks obtained from each group were further subdivided and reared under different environmental conditions from the 15th to the 42nd day as normal [N; 25 ± 1 °C, 70% relative humidity (RH)] and heat exposed (HE; 35 ± 1 °C, 50% RH) resulting in four treatment groups (CN, CHE, TCN, and TCHE). The results revealed that HSP promoter activity was stronger in CHE, which had lesser methylation and higher gene expression. The activity of promoter region was lesser in TCHE birds that were thermally manipulated at the embryonic stage, thus reflecting their stress-free condition. This was confirmed by the lower level of mRNA expression of all the HSP genes. In conclusion, thermal conditioning during embryogenesis has a positive impact and improves chicken thermotolerance capacity in postnatal life.

Sirt3 inhibits cerebral ischemia-reperfusion injury through normalizing Wnt/β-catenin pathway and blocking mitochondrial fission.

Abstract: Cerebral ischemia-reperfusion injury (IRI) potentiates existing brain damage and increases mortality and morbidity via poorly understood mechanisms. The aim of our study is to investigate the role of Sirtuin 3 (Sirt3) in the development and progression of cerebral ischemia-reperfusion injury with a focus on mitochondrial fission and the Wnt/β-catenin pathway. Our data indicated that Sirt3 was downregulated in response to cerebral IRI. However, the overexpression of Sirt3 reduced the brain infarction area and repressed IRI-mediated neuron apoptosis. Functional assays demonstrated that IRI augmented mitochondrial fission, which induced ROS overproduction, redox imbalance, mitochondrial pro-apoptotic protein leakage, and caspase-9-dependent cell death pathway activation. However, the overexpression of Sirt3 blocked mitochondrial fission and induced pro-survival signals in neurons subjected to IRI. At the molecular level, our data further illustrated that the Wnt/β-catenin pathway is required for the neuroprotection exerted by Sirt3 overexpression. Wnt/β-catenin pathway activation via inhibiting β-catenin phosphorylation attenuates mitochondrial fission and mitochondrial apoptosis. Collectively, our data show that cerebral IRI is associated with Sirt3 downregulation, Wnt/β-catenin pathway phosphorylated inactivation, and mitochondrial fission initiation, causing neurons to undergo caspase-9-dependent cell death. Based on this, strategies for enhancing Sirt3 activity and activating the Wnt/β-catenin pathway could be therapeutic targets for treating cerebral ischemia-reperfusion injury.

Differential expression of microRNAs associated with thermal stress in Frieswal (Bos taurus x Bos indicus) crossbred dairy cattle

Abstract: Environmental temperature is one of the important abiotic factors that influence the normal physiological function and productive performance of dairy cattle. Temperature stress evokes complex responses that are essential for safeguarding of cellular integrity and animal health. Post-transcriptional regulation of gene expression by miRNA plays a key role cellular stress responses. The present study investigated the differential expression of miRNA in Frieswal (Holstein Friesian × Sahiwal) crossbred dairy cattle that are distinctly adapted to environmental temperature stress as they were evolved by using the temperate dairy breed Holstein Friesian. The results indicated that there was a significant variation in the physiological and biochemical indicators estimated under summer stress. The differential expression of miRNA was observed under heat stress when compared to the normal winter season. Out of the total 420 miRNAs, 65 were differentially expressed during peak summer temperatures. Most of these miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family, and network analysis revealed most of them having stress-mediated effects on signaling mechanisms. Being greater in their expression profile during peak summer, bta-miR-2898 was chosen for reporter assay to identify its effect on the target HSPB8 (heat shock protein 22) gene in stressed bovine PBMC cell cultured model. Comprehensive understanding of the biological regulation of stress responsive mechanism is critical for developing approaches to reduce the production losses due to environmental heat stress in dairy cattle.

Characterization of a novel zinc finger transcription factor (TaZnF) from wheat conferring heat stress tolerance in Arabidopsis

Abstract: C3HC4-type zinc finger proteins are known to play important roles in various plant processes including regulation of growth and development, signaling networks, responses to abiotic stresses etc. The current study identifies and explores the involvement of TaZnF in plant stress response, mainly heat stress. TaZnF belongs to C4HC3-type zinc finger transcription factor. Phylogenetic analysis of TaZnF revealed strong sequence similarity to Brachypodium distachyon, a model system for crop species. Gene expression studies have revealed its role under diverse stress conditions including heat and cold conditions. The transcript level of TaZnF was found to be highest in seed and starts at the post anthesis period 3-5DAA, a more sensitive stage resulting in a negative influence on the yield of crop species. TaZnF possesses transcriptional activity. Overexpression of TaZnF in Arabidopsis thaliana conferred improved tolerance to both basal and high-temperature stress as observed from various assays examining their growth and development. The transgenics were recovered and showed early flowering compared to wild-type. They had larger primary roots, more lateral branching, bigger, and more numerous leaves, resulting in heavier fresh weight. Enhanced growth and early recovery resulted in bigger plants with more yield. Additionally, the overexpression Arabidopsis transgenics also showed considerable tolerance to cold and oxidative stress. These observations suggest that TaZnF acts as a positive regulator of thermal stress and thus can be of great significance in understanding and improving temperature stress tolerance in plants.

An overview of cytokines and heat shock response in polytraumatized patients

Abstract: Early after injury, local tissue damage induces a local and systemic inflammatory response that activates the immune system and leads to the development of systemic inflammatory response syndrome (SIRS). This post-traumatic response often results in uncontrolled release of inflammatory mediators and over-activation of the immune system, which occasionally results in multiple organ dysfunction syndrome (MODS). In parallel, a state of immunosuppression develops. This counter-regulating suppression of different cellular and humoral immune functions has been termed “compensatory anti-inflammatory response syndrome (CARS).” Both SIRS and CARS occur simultaneously even in the initial phase after injury. Pro- and anti-inflammatory cytokines have been suggested to play a major role in development of SIRS, although the degree of involvement of the different cytokines is quite disparate. While TNF-α and IL-1β are quite irrelevant for predicting organ dysfunction, IL-6 is the parameter that best predicts mortality. The hyperinflammatory state seems to be the cause of post-traumatic immunosuppression and heat shock proteins (HSPs), which have been proposed as one of the endogenous stimuli for the deterioration of the immune system acting as danger-associated molecular patterns (DAMPs). Extracellular HSPA1A released from injured tissues increase up to ten times immediately after trauma and even more in patients with MODS. It has powerful immune properties that could contribute to post-traumatic immunosuppression through several mechanisms that have been previously described, so HSPs could represent trauma-associated immunomodulatory mediators. For this reason, HSPA1A has been suggested to be a helpful early prognostic biomarker of trauma after severe injury: serial quantification of serum HSPA1A and anti-Hsp70 concentrations in the first hours after trauma is proposed to be used as a predictive biomarker of MODS and immunosuppression development in polytraumatized patients.

NR4A1 contributes to high-fat associated endothelial dysfunction by promoting CaMKII-Parkin-mitophagy pathways

Abstract: Parkin-related mitophagy is vital for endothelial cell viability and the development of atherosclerosis, although the upstream regulatory factor underlying Parkin-mediated mitophagy in endothelial apoptosis and atherosclerosis progression remains unknown. In the present study, we demonstrated that nuclear receptor subfamily 4 group A member 1 (NR4A1) is actually expressed in aortic endothelial cells (AECs) under oxidized low-density lipoprotein (ox-LDL) treatment in vitro or isolated from high-fat treated mice in vivo. Higher NR4A1 levels were associated with AEC apoptosis, mitochondrial dysfunction, and energy disorder. At the molecular level, ox-LDL stimulation increased NR4A1 expression, which evoked Parkin-mediated mitophagy. Excessive mitophagy overtly consumed mitochondrial mass, leading to an energy shortage and mitochondrial dysfunction. However, loss of NR4A1 protected AECs against ox-LDL induced apoptosis by inhibiting excessive mitophagy. Furthermore, we also identified that NR4A1 regulated Parkin activation via post-transcriptional modification by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII via NR4A1 induced the phosphorylated activation of Parkin. In summary, our data support the role of NR4A1/CaMKII/Parkin/mitophagy in AEC apoptosis and atherosclerosis formation and provide new insights into treating atherosclerosis with respect to endothelial viability, mitophagy, and NR4A1.

Antioxidant effect of myricitrin on hyperglycemia-induced oxidative stress in C2C12 cell.

Abstract: Hyperglycemia induced oxidative stress inside the cells. Myricitrin, as an antioxidant plant-derived component, may be useful in hyperglycemia. Hence, the aim of this study was conducted to evaluate the antioxidant effects of myricitrin on hyperglycemia-induced oxidative damage in myotubes (C2C12 cells). In this experimental study, mouse myoblast cell line (C2C12) was obtained and divided into five groups: control, hyperglycemia, hyperglycemia + myricitrin 1, 3, and 10 μM. After treatment period for 48 h, cells were collected, homogenized, and centrifuged at 2000 rpm for 10 min. All samples were kept at − 80 °C until experimental and real-time PCR assessments were performed. Hyperglycemia increased malondialdehyde (MDA) (p < 0.05), total antioxidant capacity (TAC) (p < 0.001), and cellular apoptosis, and decreased levels of superoxide dismutase (SOD), catalase (CAT) (p < 0.01), myotube glycogen content (p < 0.05), glucose transporter type 4 (Glut-4), and cellular viability (p < 0.001). Myricitrin administration improved SOD (p < 0.05), CAT (p < 0.01), muscle cell’s glycogen content (p < 0.01), Glut-4 gene expression (p < 0.001), Thiazolyl blue tetrazolium bromide (MTT) (p < 0.05), and Bax to Bcl-2 ratio (p < 0.001), and reduced MDA (p < 0.05) compared to hyperglycemia group. In conclusion, hyperglycemic condition induced oxidative stress along with cellular apoptosis, and myricitrin improved these disorders. Also, low and moderate doses of myricitrin are more efficient on skeletal muscle cells exposed to hyperglycemic statues than a high concentration of this antioxidant agent.

Modulation of Alzheimer's amyloid β peptide oligomerization and toxicity by extracellular Hsp70

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder leading to dementia caused by advanced neuronal dysfunction and death. The most significant symptoms of AD are observed at late stages of the disease when interventions are most likely too late to ameliorate the condition. Currently, the predominant theory for AD is the “amyloid hypothesis,” which states that abnormally increased levels of amyloid β (Aβ) peptides result in the production of a variety of aggregates that are neurotoxic. The specific mechanisms for Aβ peptide-induced cytotoxicity have not yet been completely elucidated. However, since the majority of Aβ is released into the extracellular milieu, it is reasonable to assume that toxicity begins outside the cells and makes its way inside where it disrupts the basic cellular process resulting in cell death. There is increasing evidence that hsp, particularly Hsp70, are exported into the extracellular milieu by an active export mechanism independent of cell death. Therefore, both Aβ peptides and Hsp70 may coexist in a common environment during pathological conditions. We observed that Hsp70 affected the Aβ assembling process in vitro preventing oligomer formation. Moreover, the presence of Hsp70 reduced the Aβ peptide-induced toxicity of cultured neurons (N2A cells). These results suggest a potential mechanism for the reduction of the detrimental effects of Aβ peptides in AD.

Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress

Abstract: Heat stress is exacerbated by global warming and affects human and animal health, leading to heart damage caused by imbalances in reactive oxygen species (ROS) and the antioxidant system, acid-base chemistry, electrolytes and respiratory alkalosis. Vitamin C scavenges excess ROS, and sodium bicarbonate maintains acid-base and electrolyte balance, and alleviates respiratory alkalosis. Herein, we explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate (Vitamin C-Na) to stimulate endogenous antioxidants and heat shock proteins (HSPs) to relieve heat stress in H9C2 cells. Control, vitamin C (20 μg/ml vitamin C for 16 h) and vitamin C-Na (20 μg/ml vitamin C-Na for 16 h) groups were heat-stressed for 1, 3 or 5 h. Granular and vacuolar degeneration, karyopyknosis and damage to nuclei and mitochondria were clearly reduced in treatment groups, as were apoptosis, lactate dehydrogenase activity and ROS and malondialdehyde levels, while superoxide dismutase activity was increased. Additionally, CRYAB, Hsp27, Hsp60 and Hsp70 mRNA levels were upregulated at 3 h (p < 0.01), and protein levels were increased for CRYAB at 0 h (p < 0.05) and 1 h (p < 0.01), and for Hsp70 at 3 and 5 h (p < 0.01). Thus, pre-treatment with vitamin C or vitamin C-Na might protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.

Isolation and characterization of human urine extracellular vesicles

Abstract: Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO4 prior to vesicle isolation by differential centrifugation. Treatment with ZnSO4 did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.

Effect of the unfolded protein response on ER protein export: a potential new mechanism to relieve ER stress.

Abstract: The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.

Retraction Note: Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways.

Abstract: Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.

Introgression of heat shock protein (Hsp70 and sHsp) genes into the Malaysian elite chilli variety Kulai ( Capsicum annuum L.) through the application of marker-assisted backcrossing (MAB)

Abstract: Backcrossing together with simple sequence repeat marker strategy was adopted to improve popular Malaysian chilli Kulai (Capsicum annuum L.) for heat tolerance. The use of molecular markers in backcross breeding and selection contributes significantly to overcoming the main drawbacks such as increase linkage drag and time consumption, in the ancient manual breeding approach (conventional), and speeds up the genome recovery of the recurrent parent. The strategy was adopted to introgress heat shock protein gene(s) from AVPP0702 (C. annuum L.), which are heat-tolerant, into the genetic profile of Kulai, a popular high-yielding chilli but which is heat sensitive. The parents were grown on seed trays, and parental screening was carried out with 252 simple sequence repeat markers. The selected parents were crossed and backcrossed to generate F1 hybrids and backcross generations. Sixty-eight markers appeared to be polymorphic and were used to assess the backcross generation; BC1F1, BC2F1 and BC3F1. The average recipient allele of the selected four BC1F1 plants was 80.75% which were used to produce the BC2F1 generation. BC1-P7 was the best BC1F1 plant because it had the highest recovery at 83.40% and was positive to Hsp-linked markers (Hsp70-u2 and AGi42). After three successive generations of backcrossing, the average genome recovery of the recurrent parent in the selected plants in BC3F1 was 95.37%. Hsp gene expression analysis was carried out on BC1F1, BC2F1 and BC3F1 selected lines. The Hsp genes were found to be up-regulated when exposed to heat treatment. The pattern of Hsp expression in the backcross generations was similar to that of the donor parent. This confirms the successful introgression of a stress-responsive gene (Hsp) into a Kulai chilli pepper variety. Furthermore, the yield performance viz. plant height, number of fruits, fruit length and weight and total yield of the improved plant were similar with the recurrent parent except that the plant height was significantly lower than the Kulai (recurrent) parent.

Role of the heat shock protein family in bone metabolism

Abstract: Heat shock proteins (HSPs) are a family of proteins produced by cells in response to exposure to stressful conditions In addition to their role as chaperones, they also play an important role in the cardiovascular, immune, and other systems Normal bone tissue is maintained by bone metabolism, particularly by the balance between osteoblasts and osteoclasts, which are physiologically regulated by multiple hormones and cytokines In recent years, studies have reported the vital role of HSPs in bone metabolism However, the conclusions remain largely controversial, and the exact mechanisms are still unclear, so a review and analyses of previous studies are of importance This article reviews the current understanding of the roles and effects of HSPs on bone cells (osteoblasts, osteoclasts, and osteocytes), in relation to bone metabolism

Alcohol exposure promotes DNA methyltransferase DNMT3A upregulation through reactive oxygen species-dependent mechanisms

Abstract: Abundant evidence has accumulated showing that fetal alcohol exposure broadly modifies DNA methylation profiles in the brain. DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, are likely implicated in this process. However, their regulation by ethanol exposure has been poorly addressed. Here, we show that alcohol exposure modulates DNMT protein levels through multiple mechanisms. Using a neural precursor cell line and primary mouse embryonic fibroblasts (MEFs), we found that ethanol exposure augments the levels of Dnmt3a, Dnmt3b, and Dnmt3l transcripts. We also unveil similar elevation of mRNA levels for other epigenetic actors upon ethanol exposure, among which the induction of lysine demethylase Kdm6a shows heat shock factor dependency. Furthermore, we show that ethanol exposure leads to specific increase in DNMT3A protein levels. This elevation not only relies on the upregulation of Dnmt3a mRNA but also depends on posttranscriptional mechanisms that are mediated by NADPH oxidase-dependent production of reactive oxygen species (ROS). Altogether, our work underlines complex regulation of epigenetic actors in response to alcohol exposure at both transcriptional and posttranscriptional levels. Notably, the upregulation of DNMT3A emerges as a prominent molecular event triggered by ethanol, driven by the generation of ROS.

Absence of cellular damage in tropical newly hatched sharks (Chiloscyllium plagiosum) under ocean acidification conditions.

Abstract: Sharks have maintained a key role in marine food webs for 400 million years and across varying physicochemical contexts, suggesting plasticity to environmental change. In this study, we investigated the biochemical effects of ocean acidification (OA) levels predicted for 2100 (pCO2 ~ 900 μatm) on newly hatched tropical whitespotted bamboo sharks (Chiloscyllium plagiosum). Specifically, we measured lipid, protein, and DNA damage levels, as well as changes in the activity of antioxidant enzymes and non-enzymatic ROS scavengers in juvenile sharks exposed to elevated CO2 for 50 days following hatching. Moreover, we also assessed the secondary oxidative stress response, i.e., heat shock response and ubiquitin levels. Newly hatched sharks appear to cope with OA-related stress through a range of tissue-specific biochemical strategies, specifically through the action of antioxidant enzymatic compounds. Our findings suggest that ROS-scavenging molecules, rather than complex enzymatic proteins, provide an effective defense mechanism in dealing with OA-elicited ROS formation. We argue that sharks' ancient antioxidant system, strongly based on non-enzymatic antioxidants (e.g., urea), may provide them with resilience towards OA, potentially beyond the tolerance of more recently evolved species, i.e., teleosts. Nevertheless, previous research has provided evidence of detrimental effects of OA (interacting with other climate-related stressors) on some aspects of shark biology. Moreover, given that long-term acclimation and adaptive potential to rapid environmental changes are yet experimentally unaccounted for, future research is warranted to accurately predict shark physiological performance under future ocean conditions.

Identification and expression analysis of multiple small heat shock protein genes in spruce budworm, Choristoneura fumiferana (L.).

Abstract: Fifteen small heat shock protein (sHSP) genes were identified from spruce budworm, Choristoneura fumiferana (L.), an important native forest pest in North America. The transcript levels of each CfHSP were measured under non-stress conditions in all life stages from egg to adult and in five different larval tissues. CfHSP transcript levels showed variation during development, with highest levels in adults and lowest in eggs. Most CfHSP transcripts are highly expressed in larval fat body and Malpighian tubules; two CfHSPs display extremely high expression in the head and epidermis. Upon heat stress, nine CfHSP genes are significantly upregulated, increasing by 50- to 2500-fold depending on developmental stage and tissue type. Upon starvation, eight CfHSPs are upregulated or downregulated, whereas six others retain constant expression. These results suggest that CfHSPs have important and multiple roles in spruce budworm development and in response to heat stress and starvation.

Ions released from a S-PRG filler induces oxidative stress in Candida albicans inhibiting its growth and pathogenicity.

Abstract: Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.