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Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin.Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and points to the presence of unknown mechanisms of Chk1 activation in mammalian cells.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

The aim of this study is to measure Advanced glycation end products (AGEs) and serum levels of Growth differentiation factor-15 (GDF-15) in regular smokers during ad-libitum (i.e., normal) smoking and following acute smoking abstinence (i.e., minimum 12 hours deprived).

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

The aim of the long-term preservation of cells, tissues and organs is to maintain their cellular structures and biological functions for as long as possible. Cryopreservation is a process where biological material is stored and preserved at very low temperatures. However, freezing and thawing processes can cause irreversible cell damage, which is related to formation of ice crystals, osmotic stress, accumulation of reactive forms of oxygen, etc. Therefore the cell viability depends mainly on the freezing rate, the composition of the cryoprotective medium as well as on the thawing rate. Using a suitable cryoprotective medium can increase the viability rate of the yeasts after “revitalization“. Appropriate pre-cultivation before freezing also plays an important role. These facts show that cell freezing and thawing processes must be controlled to avoid cell damage.

Antiviral therapies targeting the pandemic coronavirus disease 2019 (COVID-19) are urgently required. We studied an already-approved botanical drug cepharanthine (CEP) in a cell culture model of GX_P2V, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related virus. RNA-sequencing results showed the virus perturbed the expression of multiple genes including those associated with cellular stress responses such as endoplasmic reticulum (ER) stress and heat shock factor 1 (HSF1)-mediated heat shock response, of which heat shock response-related genes and pathways were at the core. CEP was potent to reverse most dysregulated genes and pathways in infected cells including ER stress/unfolded protein response and HSF1-mediated heat shock response. Additionally, single-cell transcriptomes also confirmed that genes of cellular stress responses and autophagy pathways were enriched in several peripheral blood mononuclear cells populations from COVID-19 patients. In summary, this study uncovered the transcriptome of a SARS-CoV-2-related coronavirus infection model and anti-viral activities of CEP, providing evidence for CEP as a promising therapeutic option for SARS-CoV-2 infection.

Multiple sclerosis (MS) is a central nervous system inflammatory demyelinating disease and the most common cause of non-traumatic disability in young adults. Despite progress in the treatment of the active relapsing disease, therapeutic options targeting irreversible progressive decline remain limited. Studies using skin fibroblasts derived from patients with neurodegenerative disorders demonstrate that cell stress pathways and bioenergetics are altered when compared to healthy individuals. However, findings in MS skin fibroblasts are limited. Here, we collected skin fibroblasts from 24 healthy control individuals, 30 patients with MS, and ten with amyotrophic lateral sclerosis (ALS) to investigate altered cell stress profiles. We observed endoplasmic reticulum swelling in MS skin fibroblasts, and increased gene expression of cell stress markers including BIP, ATF4, CHOP, GRP94, P53, and P21. When challenged against hydrogen peroxide, MS skin fibroblasts had reduced resiliency compared to ALS and controls. Mitochondrial and glycolytic functions were perturbed in MS skin fibroblasts while exhibiting a significant increase in lactate production over ALS and controls. Our results suggest that MS skin fibroblasts have an underlying stress phenotype, which may be disease specific. Interrogating MS skin fibroblasts may provide patient specific molecular insights and aid in prognosis, diagnosis, and therapeutic testing enhancing individualized medicine.

Oxygen is highly critical for the survival of complex organisms and lack of oxygen (hypoxia) promotes stress and death of the organism. However, cancer cells have evolved adaptive mechanisms to survive persistent pathological hypoxia experienced in the inner regions of the tumor. Normal cells rely on oxygen to meet the energy demands through mitochondrial oxidative phosphorylation, protein translation, and proper folding in the endoplasmic reticulum and for maintaining redox homeostasis. Therefore, cellular adaptive mechanisms will be employed by cancer cells through direct mechanisms such as down regulation of mitochondrial ATP production and autophagy to degrade the damage caused by hypoxia induced oxygen radicals. On the other hand, transcription factors such as hypoxia inducible factor-1 (HIF-1) and nuclear factor erythroid 2-related factor 2 (NRF2) enable the transcription of enzymes and stress responsive mechanisms to restore homeostasis indirectly. These stress response mechanisms promote resistance to cancer therapy. Hypoxia also influences other cells such as cancer stem cells (CSC) present in the hypoxic regions of the tumor. The current theory is CSCs are cells that initiate tumor with the ability to regenerate and hypoxia helps in their maintenance, thus contributes to drug resistance and relapse. Therefore, targeting hypoxia triggered adaptive mechanisms is intense area of research for drug development.

Epithelial ovarian cancer (OVCA), a fatal malignancy of women, disseminates locally. Although NK cells mount immune responses against OVCA, tumors inhibit NK cells, and the mechanism is not well understood. Cytokines stimulate NK cells; however, chronic stimulation exhausts them and induces expression of cytokine-inducible SH2-containing protein (CISH). Tumors produce anti-inflammatory cytokine interleukin (IL)-10 which may induce NK cell exhaustion. The goal of this study was to examine if CISH expression in NK cells increases during OVCA development and to determine the mechanism(s) of OVCA-induced CISH expression in NK cells. Normal ovaries (n = 7) were used for CISH, IL-10 and GRP78 expression. In tumor ovaries, CISH was examined in early and late stages (n = 14 each, all subtypes) while IL-10 and GRP78 expression were examined in early and late stage HGSC (n = 5 each). Compared to normal, the population of CISH-expressing NK cells increased and the intensity of IL-10 and GRP78 expression was significantly higher in OVCA (p < 0.05). CISH expression was positively correlated with IL-10 expression (r = 0.52, r = 0.65, p < 0.05 at early and late stages, respectively) while IL-10 expression was positively correlated with GRP78 expression (r = 0.43, r = 0.52, p < 0.05, respectively). These results suggest that OVCA development and progression are associated with increased CISH expression by NK cells which is correlated with tumor-induced persistent cellular stress.

The unfolded protein response (UPR) is a conserved and essential cellular pathway involved in protein quality control that is activated in response to several cellular stressors such as diseases states, ageing, infection and toxins. The cytosol, endoplasmic reticulum (ER) and mitochondria are continuously exposed to new proteins and in situations of aberrant protein folding; one of three lines of defence may be activated: (i) heat-shock response, (ii) mitochondrial UPR and (iii) ER UPR. These pathways lead to different signal transduction mechanisms that activate or upregulate transcription factors that, in turn, regulate genes that increase the cell's ability to correct the conformation of poorly folded proteins or, ultimately, lead to apoptosis. Despite the recent progress in understanding such biological processes, few studies have focused on the implications of the UPR in male infertility, highlighting the need for a first approach concerning the presence of these components in the male reproductive system. In testis, there is a high rate of protein synthesis, and the UPR mechanisms are well described. However, the presence of these mechanisms in spermatozoa, apparently transcriptionally inactive cells, is contentious, and it is unclear how sperm cells deal with stress. Here, we review current concepts and mechanisms of the UPR and highlight the relevance of these stress response pathways in male fertility, especially the presence and functional activation of those components in male germinal cells and spermatozoa.

Tunnelling nanotubes (TNTs) are an emerging route of long-range intercellular communication that mediate cell-to-cell exchange of cargo and organelles and contribute to maintaining cellular homeostasis by balancing diverse cellular stresses. Besides their role in intercellular communication, TNTs are implicated in several ways in health and disease. Transfer of pathogenic molecules or structures via TNTs can promote the progression of neurodegenerative diseases, cancer malignancy, and the spread of viral infection. Additionally, TNTs contribute to acquiring resistance to cancer therapy, probably via their ability to rescue cells by ameliorating various pathological stresses, such as oxidative stress, reactive oxygen species (ROS), mitochondrial dysfunction, and apoptotic stress. Moreover, mesenchymal stem cells play a crucial role in the rejuvenation of targeted cells with mitochondrial heteroplasmy and oxidative stress by transferring healthy mitochondria through TNTs. Recent research has focussed on uncovering the key regulatory molecules involved in the biogenesis of TNTs. However further work will be required to provide detailed understanding of TNT regulation. In this review, we discuss possible associations with Rho GTPases linked to oxidative stress and apoptotic signals in biogenesis pathways of TNTs and summarize how intercellular trafficking of cargo and organelles, including mitochondria, via TNTs plays a crucial role in disease progression and also in rejuvenation/therapy.

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Förster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

Diagnostics : open access journal 12(12), 3010 (2022). doi:10.3390/diagnostics12123010 special issue: "Special Issue "Biomarkers of Sepsis" / Special Issue Editor: Prof. Dr. Marcello Ciaccio, Guest Editor", Published by MDPI, Basel

Simple Summary Despite the rapid advancement in immunotherapy and targeted agents, many patients diagnosed with cancer have poor prognosis with dismal overall survival. One of the key hallmarks of cancer is the ability of cancer cells to reprogram their energy metabolism. O-GlcNAcylation is an emerging potential mechanism for cancer cells to induce proliferation and progression of tumor cells and resistance to chemotherapy. This review summarizes the mechanism behind O-GlcNAcylation and discusses the role of O-GlcNAcylation, including its function with receptor tyrosine kinase and chemo-resistance in cancer, and immune response to cancer and as a prognostic factor. Further pre-clinical studies on O-GlcNAcylation are warranted to assess the clinical efficacy of agents targeting O-GlcNAcylation. Abstract Cancer cells are able to reprogram their glucose metabolism and retain energy via glycolysis even under aerobic conditions. They activate the hexosamine biosynthetic pathway (HBP), and the complex interplay of O-linked N-acetylglucosaminylation (O-GlcNAcylation) via deprivation of nutrients or increase in cellular stress results in the proliferation, progression, and metastasis of cancer cells. Notably, cancer is one of the emerging diseases associated with O-GlcNAcylation. In this review, we summarize studies that delineate the role of O-GlcNAcylation in cancer, including its modulation in metastasis, function with receptor tyrosine kinases, and resistance to chemotherapeutic agents, such as cisplatin. In addition, we discuss the function of O-GlcNAcylation in eliciting immune responses associated with immune surveillance in the tumor microenvironment. O-GlcNAcylation is increasingly accepted as one of the key players involved in the activation and differentiation of T cells and macrophages. Finally, we discuss the prognostic role of O-GlcNAcylation and potential therapeutic agents such as O-linked β-N-acetylglucosamine-transferase inhibitors, which may help overcome the resistance mechanism associated with the reprogramming of glucose metabolism.

The interaction of heat shock proteins (HSP) with cellular membranes has been an enigmatic process, initially observed by morphological studies, inferred during the purification of HSP70s, and confirmed after the detection of these proteins on the surface of cancer cells and their insertion into artificial lipid bilayers. Today, the association of several HSP with lipid membranes is well established. However, the mechanisms for membrane insertion have been elusive. There is conclusive evidence indicating that HSP70s have a great selectivity for negatively charged phospholipids, whereas other HSP have a broader spectrum of lipid specificity. HSP70 also oligomerizes upon membrane insertion, forming ion conductance channels. The functional role of HSP70 lipid interactions appears related to membrane stabilization that may play a role during cell membrane biogenesis. They could also play a role as membrane chaperones as well as during endocytosis, microautophagy, and signal transduction. Moreover, HSP membrane association is a key component in the extracellular export of these proteins. The presence of HSP70 on the surface of cancer cells and its interaction with lysosome membranes have been envisioned as potential therapeutic targets. Thus, the biology and function of HSP membrane association are reaching a new level of excitement. This review is an attempt to preserve the recollection of the pioneering contributions of many investigators that have participated in this endeavor.

Most eukaryotic genes harbor multiple cleavage and polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms. APA regulation has been implicated in a diverse array of physiological and pathological conditions. While RNA sequencing tools that generate reads containing the PAS, named onSite reads, have been instrumental in identifying PASs, they have not been widely used. By contrast, a growing number of methods generate reads that are close to the PAS, named nearSite reads, including the 3’ end counting strategy commonly used in single cell analysis. How these nearSite reads can be used for APA analysis, however, is poorly studied. Here, we present a computational method, named model-based analysis of alternative polyadenylation using 3’ end-linked reads (MAAPER), to examine APA using nearSite reads. MAAPER uses a probabilistic model to predict PASs for nearSite reads with high accuracy and sensitivity, and examines different types of APA events, including those in 3’UTRs and introns, with robust statistics. We show MAAPER’s accuracy with data from both bulk and single cell RNA samples and its applicability in unpaired or paired experimental designs. Our result also highlights the importance of using well annotated PASs for nearSite read analysis.

Els fibroblasts cardíacs són els principals productors de la matriu extracel·lular que cohesiona la musculatura del cor, conferint-li les propietats ideals per transformar la contracció dels cardiomiòcits en el moviment de la paret cardíaca que permet el bombeig de la sang. A més, aquestes cèl·lules intervenen en el procés de sincronització del batec i la secreció de factors tròfics potenciant el creixement del cor durant el desenvolupament. En condicions patològiques, com ara el tall del flux sanguini al miocardi (isquèmia), els fibroblasts cardíacs secreten la matriu que generarà la cicatriu durant la mort del miocardi. No obstant, tot i que aquest esdeveniment és essencial per sobreviure a l’infart, la deposició de matriu extracel·lular pot representar una complicació si s’allarga en el temps o s’activa excessivament. Per això, s’està destinant molt esforç a la identificació de l’origen embrional i els tipus cel·lulars precisos que constitueixen la població de fibroblasts cardíacs, a re-avaluar la seva abundància al cor, així com a comprendre la regulació de la síntesi i secreció de matriu extracel·lular. El nostre grup havia demostrat que els fibroblasts cardíacs expressen elevats nivells de Bcl-2, una proteïna anti-apoptòtica que els protegeix la integritat mitocondrial durant la isquèmia, reduint l’activació de les caspases. Tot i que estan ben caracteritzats pel que fa a la quantitat, l’origen genètic i l’activitat dels fibroblasts cardíacs, hi ha poca informació sobre els mecanismes implicats en la resistència d’aquestes cèl·lules a la isquèmia i per això, vam decidir seguir investigant aquest aspecte. La hipòtesi inicial era que l’autofàgia, un procés implicat en el reciclatge de components cel·lulars regulat per Bcl-2 i que pot ser utilitzat per obtenir energia, podia estar implicada en la major supervivència dels fibroblasts cardíacs a la isquèmia. L’anàlisi de l’autofàgia a fibroblasts primaris neonatals de rata, utilitzant tant inhibidors químics com el silenciament de gens clau, va mostrar que és un procés important en condicions normals però no durant la isquèmia en aquestes cèl·lules. Malgrat això, vam trobar que l’elevada expressió de Bcl-2 és necessària per la supervivència dels fibroblasts cardíacs en condicions normals, a més de durant la isquèmia i l’estrès de reticle. També vam trobar que aquestes cèl·lules presenten diverses característiques associades a la major capacitat de supervivència en relació a d’altres fibroblasts, incloent major respiració basal i de reserva, nivells de radicals lliures d’oxigen elevats però millor controlats durant la isquèmia, nivells elevats d’expressió d’enzims antioxidants i de complexos de la cadena respiratòria, però menys expressió de Pgc-1α i Mitofusina-2, associat a una xarxa mitocondrial amb més mitocondris aïllats. Aquestes característiques es troben relacionades amb la major activació de senyalització inflamatòria i vam mostrar que moltes depenen d’una activació basal elevada de la via de transducció de senyal Jak/Stat. En conjunt, els nostres resultats ajuden a la millor comprensió de les característiques biològiques subjacents a la major capacitat de supervivència dels fibroblasts cardíacs. Los fibroblastos cardíacos son los principales productores de la matriz extracelular que cohesiona la musculatura del corazón, confiriéndole las propiedades ideales para transformar la contracción de los cardiomiocitos en el movimiento de la pared cardíaca que permite el bombeo de la sangre. Además, estas células intervienen en el proceso de sincronización del latido y la secreción de factores tróficos potenciando el crecimiento del corazón durante el desarrollo. En condiciones patológicas, tales como el corte del flujo sanguíneo al miocardio (isquemia), los fibroblastos cardíacos secretan la matriz que generará la cicatriz durante la muerte del miocardio. Sin embargo, aunque este evento es esencial para sobrevivir al infarto, la deposición de matriz extracelular puede representar una complicación si se alarga en el tiempo o se activa excesivamente. Debido a eso, se está destinando mucho esfuerzo a la identificación del origen embrionario y los tipos celulares precisos que constituyen la población de fibroblastos cardíacos, a re-evaluar su abundancia en el corazón, así como a comprender la regulación de la síntesis y secreción de matriz extracelular. Nuestro grupo había demostrado que los fibroblastos cardíacos expresan niveles elevados de Bcl-2, una proteína anti-apoptótica que protege la integridad mitocondrial durante la isquemia, reduciendo la activación de las caspasas. Aunque se han caracterizado bien la cantidad, el origen genético y la actividad de los fibroblastos cardíacos, hay poca información sobre los mecanismos implicados en la resistencia a la isquemia de estas células y por eso decidimos seguir investigando este aspecto. La hipótesis inicial era que la autofagia, un proceso implicado en el reciclaje de componentes celulares regulado por Bcl-2 y que puede ser utilizado para obtener energía, podía estar implicada en la mayor supervivencia de los fibroblastos cardíacos a la isquemia. El análisis de la autofagia en fibroblastos primarios neonatales de rata, utilizando tanto inhibidores químicos como el silenciamiento de genes clave, mostró que es un proceso importante en condiciones normales, pero no durante la isquemia en estas células. Sin embargo, encontramos que la elevada expresión de Bcl-2 es necesaria para la supervivencia de los fibroblastos cardíacos en condiciones normales, además de durante la isquemia y el estrés de retículo. Encontramos también que estas células presentan varias características asociadas a la mayor capacidad de supervivencia en relación a otros fibroblastos, incluyendo mayor respiración basal y de reserva, niveles de radicales libres de oxígeno elevados pero mejor controlados durante la isquemia, niveles elevados de expresión de enzimas antioxidantes y de complejos de la cadena respiratoria, pero menos expresión de Pgc-1α y Mitofusina-2, asociado a una red mitocondrial con más mitocondrias aisladas. Estas características están relacionadas con la mayor activación de señalización inflamatoria y mostramos que muchas dependen de una activación basal elevada de la vía de transducción de señal Jak/Stat. En conjunto, nuestros resultados ayudan a una mejor comprensión de las características biológicas subyacentes a la mayor capacidad de supervivencia de los fibroblastos cardíacos. Cardiac fibroblasts are the main producers of the extracellular matrix that binds together the cardiac muscle, giving it the ideal properties to transform the contraction of cardiomyocytes into the movement of the heart wall that allows blood pumping. In addition, these cells intervene in the process of beat synchronization and the secretion of trophic factors, enhancing the growth of the heart during development. In pathological conditions such as cut off of blood flow to the myocardium (ischemia), cardiac fibroblasts secrete the matrix that will generate the scar during myocardial death. Although this event is essential to survive the infarction, the deposition of extracellular matrix can represent a complication if it is prolonged in time or is excessively activated. That is why a lot of effort is being devoted to identifying the embryonic origin and the precise cell types that make up the population of cardiac fibroblasts, to re-evaluating their abundance in the heart, as well as to understanding the regulation of extracellular matrix synthesis and secretion. Our group had shown that cardiac fibroblasts express a high level of Bcl-2, an anti-apoptotic protein that protects mitochondrial integrity during ischemia, reducing the activation of caspases. Although the number, genetic origin, and activity of cardiac fibroblasts have been well characterized, there is little information on the mechanisms involved in the resistance of these cells to ischemia and that is why we decided to continue investigating this aspect. The initial hypothesis was that autophagy, a process involved in the recycling of cellular components regulated by Bcl-2 and that can be used to obtain energy, could be involved in the greater survival of cardiac fibroblasts to ischemia. Analysis of autophagy in neonatal rat primary fibroblasts, using both chemical inhibitors and key gene silencing, showed that it is an important process under normal conditions, but not during ischemia in these cells. However, we found that the high expression of Bcl-2 is necessary for the survival of cardiac fibroblasts under normal conditions, as well as during ischemia and reticulum stress. We also found that these cells present several characteristics associated with a higher survival capacity in relation to other fibroblasts, including greater basal and reserve respiration, elevated levels of reactive oxygen species, which are better controlled during ischemia, high levels of expression of antioxidant enzymes and of complexes of the respiratory chain, but less expression of Pgc-1α and Mitofusin-2, associated with a mitochondrial network with more isolated mitochondria. These characteristics are related to the greater activation of inflammatory signaling and we show that many of them depend on a high basal activation of the Jak/Stat signal transduction pathway. Taken together, our results help to better understand the biological characteristics underlying the greater survival capacity of cardiac fibroblasts.

The nuclear factor E2-related factor 1 (Nrf1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of Nrf1 proteins, but the regulatory enzymes that act on Nrf1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of Nrf1 activity. We found that USP7 interacts with Nrf1a and TCF11-the two long protein isoforms of Nrf1. Expression of wildtype USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and Nrf1a, leading to their increased stability and increased transactivation of reporter gene expression by TCF11 and Nrf1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and Nrf1a and reduction of their steady state levels. Loss of USP7 function attenuated the induction of Nrf1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of Nrf1 protein to induce gene expression in response to toxic metal exposure.

The ability§ of the heart to adapt to changes in the mechanical environment is critical for normal cardiac physiology. The role of nitric oxide is increasingly recognized as a mediator of mechanical signaling. Produced in the heart by nitric oxide synthases, nitric oxide affects almost all mechano-transduction pathways within the cardiomyocyte, with roles mediating mechano-sensing, mechano-electric feedback (via modulation of ion channel activity), and calcium handling. As more precise experimental techniques for applying mechanical stresses to cells are developed, the role of these forces in cardiomyocyte function can be further understood. Furthermore, specific inhibitors of different nitric oxide synthase isoforms are now available to elucidate the role of these enzymes in mediating mechano-electrical signaling. Understanding of the links between nitric oxide production and mechano-electrical signaling is incomplete, particularly whether mechanically sensitive ion channels are regulated by nitric oxide, and how this affects the cardiac action potential. This is of particular relevance to conditions such as atrial fibrillation and heart failure, in which nitric oxide production is reduced. Dysfunction of the nitric oxide/mechano-electrical signaling pathways are likely to be a feature of cardiac pathology (e.g., atrial fibrillation, cardiomyopathy, and heart failure) and a better understanding of the importance of nitric oxide signaling and its links to mechanical regulation of heart function may advance our understanding of these conditions.

Chronic stress is a combination of nonspecific adaptive reactions of the body to the influence of various adverse stress factors which disrupt its homeostasis, and it is also a corresponding state of the organism’s nervous system (or the body in general). We hypothesized that chronic stress may be one of the causes occurence of several molecular and cellular types of stress. We analyzed literary sources and considered most of these types of stress in our review article. We examined genes and mutations of nuclear and mitochondrial genomes and also molecular variants which lead to various types of stress. The end result of chronic stress can be metabolic disturbance in humans and animals, leading to accumulation of reactive oxygen species (ROS), oxidative stress, energy deficiency in cells (due to a decrease in ATP synthesis) and mitochondrial dysfunction. These changes can last for the lifetime and lead to severe pathologies, including neurodegenerative diseases and atherosclerosis. The analysis of literature allowed us to conclude that under the influence of chronic stress, metabolism in the human body can be disrupted, mutations of the mitochondrial and nuclear genome and dysfunction of cells and their compartments can occur. As a result of these processes, oxidative, genotoxic, and cellular stress can occur. Therefore, chronic stress can be one of the causes forthe occurrence and development of neurodegenerative diseases and atherosclerosis. In particular, chronic stress can play a large role in the occurrence and development of oxidative, genotoxic, and cellular types of stress.

Background Cellular sex has rarely been considered as a biological variable in preclinical research, even when the pathogenesis of diseases with predictable sex differences is studied. In this perspective, proteomics, and “omics” approaches in general, can provide powerful tools to obtain comprehensive cellular maps, thus favoring the discovery of still unknown sex-biased physio-pathological mechanisms. Methods We performed proteomic and Gene Ontology (GO) analyses of the secretome from human serum-deprived male and female endothelial cells (ECs) followed by ELISA validation. Apoptosis was detected by FACS and Western blot techniques and efferocytosis through the ability of the macrophage cell line RAW 264.7 to engulf apoptotic ECs. PTX3 mRNA levels were measured by RT-qPCR. Results Proteomic and GO analyses of the secretome from starved human male and female ECs demonstrated a significant enrichment in proteins related to cellular responses to stress and to the regulation of apoptosis in the secretome of male ECs. Accordingly, a higher percentage of male ECs underwent apoptosis in response to serum deprivation in comparison with female ECs. Among the secreted proteins, we reliably found higher levels of PTX3 in the male EC secretome. The silencing of PTX3 suggested that male ECs were dependent on its expression to properly carry out the efferocytotic process. At variance, female EC efferocytosis seemed to be independent on PTX3 expression. Conclusions Our results demonstrated that serum-starved male and female ECs possess different secretory phenotypes that might take part in the sex-biased response to cellular stress. We identified PTX3 as a crucial player in the male-specific endothelial response to an apoptotic trigger. This novel and sex-related role for secreted proteins, and mainly for PTX3, may open the way to the discovery of still unknown sex-specific mechanisms and pharmacological targets for the prevention and treatment of endothelial dysfunction at the onset of atherosclerosis and cardiovascular disease.

The review aims to consolidate research findings on the molecular mechanisms and virulence and pathogenicity characteristics of coronavirus disease (COVID-19) causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and their relevance to four typical stages in the development of acute viral infection. These four stages are invasion; primary blockade of antiviral innate immunity; engagement of the virus’s protection mechanisms against the factors of adaptive immunity; and acute, long-term complications of COVID-19. The invasion stage entails the recognition of the spike protein (S) of SARS-CoV-2 target cell receptors, namely, the main receptor (angiotensin-converting enzyme 2, ACE2), its coreceptors, and potential alternative receptors. The presence of a diverse repertoire of receptors allows SARS-CoV-2 to infect various types of cells, including those not expressing ACE2. During the second stage, the majority of the polyfunctional structural, non-structural, and extra proteins SARS-CoV-2 synthesizes in infected cells are involved in the primary blockage of antiviral innate immunity. A high degree of redundancy and systemic action characterizing these pathogenic factors allows SARS-CoV-2 to overcome antiviral mechanisms at the initial stages of invasion. The third stage includes passive and active protection of the virus from factors of adaptive immunity, overcoming of the barrier function at the focus of inflammation, and generalization of SARS-CoV-2 in the body. The fourth stage is associated with the deployment of variants of acute and long-term complications of COVID-19. SARS-CoV-2’s ability to induce autoimmune and autoinflammatory pathways of tissue invasion and development of both immunosuppressive and hyperergic mechanisms of systemic inflammation is critical at this stage of infection.

ONC206 (Oncoceutics) is an imipiridone with nanomolar potency and analogue of ONC201, a selective dopamine receptor D2 (DRD2) antagonist currently being investigated in phase II clinical trials for serous endometrial cancer (SEC). This study investigated the anti-proliferative efficacy of ONC206 in SEC cell lines as well as its impact on cellular stress and adhesion/invasion. ONC206 inhibited cellular proliferation in a dose-dependent manner and was more potent than ONC201 in the ARK1 (IC50 = 0.33µM vs. IC50 = 1.59uM) and SPEC-2 (IC50 = 0.24uM vs. IC50 = 0.81uM) cell lines. Treatment with ONC206 resulted in induction of ROS production and reduction of mitochondrial membrane potential, accompanied by an increase in cleaved caspase-3 and caspase-9 activity (p < 0.01). ONC206 also significantly inhibited cellular adhesion and migration in both cell lines (p < 0.01). Pretreatment with the stress inhibitor N-acetylcysteine (NAC) significantly attenuated the efficacy of ONC206 on cell proliferation, ROS production and cellular invasion. ONC206 demonstrates nanomolar potency for the inhibition of proliferation in SEC cells. Specifically, ONC206 utilizes ISR activation as a significant pathway in the propagation of its anti-proliferative and anti-metastatic effects. Thus, ONC206 may be a promising agent in future SEC clinical trials as was its predecessor ONC201.

Simple Summary Prostate cancer cells produce lipids via the activation of a specific pathway called fatty acid synthesis, also known as De novo lipogenesis. This pathway is essential for the survival and growth of most types of cancer cells, including prostate cancer. In our study, we showed that prostate cancer cells activate this lipid synthesis pathway to become more aggressive and develop resistance to commonly used therapeutic agents for advanced prostate cancer such as enzalutamide, an effective and commonly used androgen receptor (AR) targeted agent. Interestingly, by combining enzalutamide with a lipid synthesis pathway inhibitor, we were able to show that growth of prostate cancer tumors was more effectively reduced than with either agent alone. We also showed that this combination led to cell stress and death by changing the lipid content in the cell. These important findings could lead to new therapeutic strategies combining effective AR targeted therapies with lipid synthesis inhibitors for the treatment of advanced prostate cancer. Abstract De novo lipogenesis (DNL) is now considered as a hallmark of cancer. The overexpression of key enzymes of DNL is characteristic of both primary and advanced disease and may play an important role in resistance to therapies. Here, we showed that DNL is highly enhanced in castrate resistant prostate cancer (CRPC) cells compared to hormone sensitive and enzalutamide resistant cells. This observation suggests that this pathway plays an important role in the initiation of aggressive prostate cancer and in the development of enzalutamide resistance. Importantly, here we show that both prostate cancer cells sensitive and resistant to enzalutamide are dependent on DNL to proliferate. We next combined enzalutamide with an inhibitor of Stearoyl CoA Desaturase 1 (SCD1), an important enzyme in DNL, and observed significantly reduced tumor growth caused by the important change in tumoral lipid desaturation. Our findings suggest that the equilibrium between monounsaturated fatty acids and saturated fatty acids is essential in the establishment of the more aggressive prostate cancer phenotype and that the combination therapy induces a disruption of this equilibrium leading to an important decrease of cell proliferation. These findings provide new insights into the role of DNL in the progression of prostate cancer cells. The study also provides the rationale for the use of an inhibitor of SCD1 in combination with enzalutamide to improve response, delay enzalutamide resistance and improve disease free progression.

Lavandula angustifolia Mill. (lavender) and Origanum heracleoticum L. (greek oregano) are aromatic plants used in traditional medicine for relief of convulsion, anxiety, insomnia and in the treatment of neurological disorders. On the basis of literature, we evaluated the changing in glutathione enzymatic activities provoked by the essential oils and pure components, linalool, carvacrol and limonene to study the mechanisms of action, responsible for several activities. Activities of SOD (superoxide dismutase) and GPx (glutathione peroxidase) tend to increase respect to control. SOD maximal increase reached up to 117% for carvacrol and limonene. Increase in GPx activity reached even up to value 1.229 for origanum oil in comparison to control 0.125 μkat/mg prot. Activities of GR (glutathione reductase), except for lavender, showed a biphasic response. Like for GR, the administration of compounds, resulted in a biphasic response in GST (glutathione-S-transferase) activities (with a consistent increase in activity at concentration 125 μg/mL for all compounds except lavender). Moreover, the changes in GSH (reduced glutathione), are no significant for different concentrations of essential oils. So, the biological properties of essential oils and specifically, the antioxidant ones, can be related to their capacity to modify the glutathione enzymatic activities.

Measures of microbial growth, used as indicators of cellular stress, are sometimes quantified at a single time-point. In reality, these measurements are compound representations of length of lag, exponential growth-rate, and other factors. Here, we investigate whether length of lag phase can act as a proxy for stress, using a number of model systems (Aspergillus penicillioides; Bacillus subtilis; Escherichia coli; Eurotium amstelodami, E. echinulatum, E. halophilicum, and E. repens; Mrakia frigida; Saccharomyces cerevisiae; Xerochrysium xerophilum; Xeromyces bisporus) exposed to mechanistically distinct types of cellular stress including low water activity, other solute-induced stresses, and dehydration-rehydration cycles. Lag phase was neither proportional to germination rate for X. bisporus (FRR3443) in glycerol-supplemented media (r2 = 0.012), nor to exponential growth-rates for other microbes. In some cases, growth-rates varied greatly with stressor concentration even when lag remained constant. By contrast, there were strong correlations for B. subtilis in media supplemented with polyethylene-glycol 6000 or 600 (r2 = 0.925 and 0.961), and for other microbial species. We also analysed data from independent studies of food-spoilage fungi under glycerol stress (Aspergillus aculeatinus and A. sclerotiicarbonarius); mesophilic/psychrotolerant bacteria under diverse, solute-induced stresses (Brochothrix thermosphacta, Enterococcus faecalis, Pseudomonas fluorescens, Salmonella typhimurium, Staphylococcus aureus); and fungal enzymes under acid-stress (Terfezia claveryi lipoxygenase and Agaricus bisporus tyrosinase). These datasets also exhibited diversity, with some strong- and moderate correlations between length of lag and exponential growth-rates; and sometimes none. In conclusion, lag phase is not a reliable measure of stress because length of lag and growth-rate inhibition are sometimes highly correlated, and sometimes not at all.

publishedVersion

A key element of cellular homeostasis lies in the way in which misfolded and dysfunctional proteins are handled. Cellular pathways that include proteasomal destruction and autophagic disposal are components of normal proteostasis. Here we report a novel molecule that plays a non-redundant role in maintaining homeostasis, Fas Apoptosis Inhibitory Molecule (FAIM). FAIM is highly conserved throughout evolution and bears no homology to any other protein. We found that FAIM counteracts heat and oxidative stress-induced loss of cell viability. FAIM is recruited to ubiquitinated proteins induced by cellular stress and the levels of stress-induced protein aggregates are much greater in FAIM-deficient cell lines. Primary fibroblasts from FAIM-deficient mice showed the same proteostasis deficits as cell lines. Administration of a mediator of oxidative stress to FAIM-deficient animals induced more ubiquitinated protein aggregates and more organ damage as compared to wild type mice. These results identify a completely new actor that protects cells against stress-induced loss of viability by preventing protein aggregation.

Induced by overfeeding, hepatic steatosis is a process exploited for the “foie gras” production in mule ducks. To better understand the mechanisms underlying its development, the physiological responses of mule ducks overfed with corn for a duration of 11 days were analyzed. A kinetic analysis of glucose and lipid metabolism and cell protection mechanisms was performed on 96 male mule ducks during overfeeding with three sampling times (after the 4th, the 12th, and the 22nd meal). Gene expression and protein analysis realized on the liver, muscle, and abdominal fat showed an activation of a cholesterol biosynthetic pathway during the complete overfeeding period mainly in livers with significant correlations between its weight and its cholesterolemia ( r = 0.88; P < 0.0001) and between the liver weight and the hmgcr and soat1 expression ( r = 0.4, P < 0.0001 and r = 0.67; P < 0.0001, respectively). Results also revealed an activation of insulin and amino acid cells signaling a pathway suggesting that ducks boost insulin sensitivity to raise glucose uptake and use via glycolysis and lipogenesis. Cellular stress analysis revealed an upregulation of key autophagy-related gene expression atg8 and sqstm1( P < 0.0001) during the complete overfeeding period, mainly in the liver, in contrast to an induction of cyp2e1( P < 0.0001), suggesting that autophagy could be suppressed during steatosis development. This study has highlighted different mechanisms enabling mule ducks to efficiently handle the starch overload by keeping its liver in a nonpathological state. Moreover, it has revealed potential biomarker candidates of hepatic steatosis as plasma cholesterol for the liver weight.

Cellular stress has long been acknowledged as a component of disease. More recently, several cellular stress related exosome and lncRNA-based functional mechanisms have been identified in two different disease models. Importantly, the thesis concludes by investigating methods to address and modulate cellular stress as a possible therapeutic treatment to disease. In this project, the cooperative functional role of lncRNAs and exosomes in two disease models was investigated. In chapter 1, we hypothesised that exosomal lncRNAs can modulate cellular signalling and micro-environments in cancer. This was extended by exploring the relationship between exosomes and lncRNAs and the disease cystic fibrosis in chapter 2. Cellular stress and the potential impact of exosomes and lncRNAs on the disease at the cellular level served as the connection between the two different disease models. Our experiments in chapter 3 highlighted the functional capacity of lncRNAs to modulate cellular stress in disease states. Using transcriptome profiling of heat shocked cells, I discovered that the lncRNA retro-EIF2S2 has the ability to modulate cell adherence in response to heat shock. Interestingly, I then demonstrated that exosomes isolated from cancer cells can transfer functional lncRNAs to their recipient cells in chapter 4. Examination of these cancer cell exosomes identified several highly expressed exosomal lncRNA sequences that modulated cell viability. The ubiquity of modulating cellular stress via exosomal transfer in diseased cells was confirmed in cells isolated from CF patients in chapter 5. In these experiments, using primary patient epithelial cells and exosomes, I demonstrate that serum-derived exosomes induce NETosis, modulate wound healing and up regulate inflammation. The potential for moderating cellular stress as a therapeutic strategy was explored in the final experimental chapter (chapter 6). In this approach, I create an oxidative stress model of CF by challenging differentiated bronchial epithelial cells with the bacterial endotoxin lipopolysaccharide and I test a novel antioxidant compound to evaluate its efficacy in resolving cellular stress. Collectively, these results demonstrate the interconnectivity between various modes of cellular stress and disease while highlighting the importance of treating cellular stress when investigating possible avenues of disease treatment.

Mitochondrial bioenergetics require the coordination of two different and independent genomes. Mutations in either genome will affect mitochondrial functionality and produce different sources of cellular stress. Depending on the kind of defect and stress, different tissues and organs will be affected, leading to diverse pathological conditions. There is no curative therapy for mitochondrial diseases, nevertheless, there are strategies described that fight the various stress forms caused by the malfunctioning organelles. Here, we will revise the main kinds of stress generated by mutations in mitochondrial genes and outline several ways of fighting this stress.

Background:Aluminium salts have been used for decades in vaccines as adjuvants to facilitate the adaptive immune response against co-administered antigens. Two types of aluminium adjuvant are mostly used, aluminium oxyhydroxide and aluminium hydroxyphosphate. Both types of aluminium adjuvant consist of nanoparticles that form loose, micrometre sized aggregates at circumneutral pH.Aluminium adjuvants constitute a well-documented example of administration of nanomaterials to humans with infrequent side effects and a safety record generally regarded as excellent. However, despite its prolonged use in human and veterinary medicine, the mechanisms behind the enhanced response and the immune stimulatory effect are still by and large unknown.Methods:The present paper reviews existing ideas regarding the immunostimulatory effects of aluminium adjuvants, with a focus on the induction of an inflammatory response by cellular stress. Reviewed information was obtained from peer-reviewed scientific papers published in 1988 to date with one exception, a paper published 1931.Results:Cellular stress causes extra cellular signalling of Danger Associated Molecular Patterns (DAMPs) and upon phagocytosis of aluminium adjuvants the cells need to manage the ingested particles.Conclusion:A persistent intracellular accumulation of aluminium adjuvants will be a solid depository of sparingly soluble aluminium salts maintaining a constant concentration of Al3+ions in the cytoplasm and this will affect multiple biochemical processes. The cell will be under constant stress and DAMP signalling will occur and we would like to suggest the maintenance of a constant concentration Al3+ions in the cytoplasm as a general underlying feature of the immune stimulation properties of aluminium adjuvants.

The concept of bladder ischemia as a contributing factor to detrusor overactivity and lower urinary tract symptoms (LUTS) is evolving. Bladder ischemia as a consequence of pelvic arterial atherosclerosis was first documented in experimental models and later in elderly patients with LUTS. It was shown that early-stage moderate ischemia produces detrusor overactivity, while prolonged severe ischemia provokes changes consistent with detrusor underactivity. Recent studies imply a central role of cellular energy sensors, cellular stress sensors, and stress response molecules in bladder responses to ischemia. The cellular energy sensor adenosine monophosphate-activated protein kinase was shown to play a role in detrusor overactivity and neurodegeneration in bladder ischemia. The cellular stress sensors apoptosis signal-regulating kinase 1 and caspase-3 along with heat shock proteins were characterized as important contributing factors to smooth muscle structural modifications and apoptotic responses in bladder ischemia. Downstream pathways seem to involve hypoxia-inducible factor, transforming growth factor beta, vascular endothelial growth factor, and nerve growth factor. Molecular responses to bladder ischemia were associated with differential protein expression, the accumulation of non-coded amino acids, and post-translational modifications of contractile proteins and stress response molecules. Further insight into cellular stress responses in bladder ischemia may provide novel diagnostic and therapeutic targets against LUTS.

In farmed animals, personalities have demonstrated links to performance traits, health and welfare, disease susceptibility, and to coping with environmental stress. The teleost caudal neurosecretory system (CNSS) of the posterior spinal cord is involved in the regulation of the osmoregulatory and stress responses and can directly sense a hypothermal challenge via a transient receptor potential ion channel. Here we investigated the global transcriptomic response of the CNSS of shy and bold individuals of the olive flounder (Paralichthys olivaceus) acclimated to 18 degrees C and transferred to 12 degrees C (low temperature treatment, LT) or 24 degrees C (high temperature treatment, HT) for 2 h (acute) or 8 days (chronic) in relation to fish maintained at 18 degrees C (control). Overall, differential transcriptomic responses were stronger in bold individuals and induced by acute HT and chronic LT treatments. Acute HT induced innate immunity, circadian rhythm, and cellular stress response pathways and, specifically in bold individuals, a heightened response of molecular chaperones of cellular response to heat which may be related to the mechanisms of rescue of downregulated RNA splicing processes. Chronic LT also caused downregulation of innate immunity pathways as well as pathways related to cell division. In addition, the CNSS of bold individuals was enriched in pathways related to regulation of cell cycle, adaptive immune response, and apoptosis, while cellular metabolism pathways were downregulated in shy individuals. The putative temperature sensor genes Trpv1 and Trpa1 were upregulated, respectively, after acute HT and chronic LT treatments, but there was no indication of a specific neurosecretory response of the CNSS. While several of the observed transcriptomic responses to temperature appear to be shared by other tissues and species, it is also clear that bold and shy behavioural personalities show marked different responses, with bold individuals the most responsive. These results add to the knowledge base available on olive flounder aquaculture, indicating a differential role of the CNSS in the response to temperature change according to behavioural personalities. National Natural Science Foundation of China (NSFC) 31572599 41376134; China Scholarship Council; Portuguese Foundation for Science and Technology: UIDB/04326/2020. info:eu-repo/semantics/publishedVersion

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5' end of the mRNA and scans the 5' untranslated region (5'UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5'UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N 6-methyladenosine (m6A) residues in their 5'UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets. This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). Rafaela Lacerda and Juliane Menezes were supported by fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BD/74778/2010 and SFRH/BPD/98360/2013, respectively). info:eu-repo/semantics/publishedVersion

Cells of an organism face with various types of insults during their lifetime. Exposure to toxins, metabolic problems, ischaemia/reperfusion, physical trauma, genetic diseases, neurodegenerative diseases are among the conditions that trigger cellular stress responses. In this context, autophagy is one of the mechanisms that supports cell survival under stressful conditions. Autophagic vesicle engulfs the cargo and transports it to lysosome for degradation and turnover. As such, autophagy eliminates abnormal proteins, clears damaged organelles, limits oxidative stress and helps to improve metabolic balance. Nervous system cells and particularly postmitotic neurons are highly sensitive to a spectrum of insults, and autophagy emerges as one of the key stress response mechanism, ensuring health and survival of these vulnerable cell types. In this review, we will overview mechanisms through which cells cope with stress, and how these stress responses regulate autophagy, with a special focus on the nervous system. (C) 2020 Elsevier Ltd. All rights reserved.

Objectives: The occurrence of oxidative stress and endoplasmic reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated and play an important role in liver injury. During viral infection, hepatocytes must handle not only the replication of the virus, but also inflammatory signals generating oxidative stress and damage. Although several mechanisms exist to overcome cellular stress, little attention has been given to the adaptive response of hepatocytes during exposure to multiple noxious triggers. Methods: In the present study, Huh-7 cells and hepatocytes expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER stress, were additionally challenged with the superoxide anion generator menadione to mimic external oxidative stress. The production of reactive oxygen species (ROS) as well as the response to oxidative stress and ER stress were investigated. Results: We demonstrate that hepatocytes diminish oxidative stress through a reduction in ROS production, ER-stress markers (HSPA5 [GRP78], sXBP1) and apoptosis (caspase-3 activity) despite external oxidative stress. Interestingly, the level of the autophagy substrate protein p62 was downregulated together with HCV Core degradation, suggesting that hepatocytes can overcome excess oxidative stress through autophagic degradation of one of the stressors, thereby increasing cell survival. Duscussion: In conclusion, hepatocytes exposed to direct and indirect oxidative stress inducers are able to cope with cellular stress associated with viral hepatitis and thus promote cell survival.

Dysregulation of protein translation is emerging as a unifying mechanism in the pathogenesis of many neuronal disorders. Ribosomal RNA (rRNA) and transfer RNA (tRNA) are structural molecules that have complementary and coordinated functions in protein synthesis. Defects in both rRNAs and tRNAs have been described in mammalian brain development, neurological syndromes, and neurodegeneration. In this review, we present the molecular mechanisms that link aberrant rRNA and tRNA transcription, processing and modifications to translation deficits, and neuropathogenesis. We also discuss the interdependence of rRNA and tRNA biosynthesis and how their metabolism brings together proteotoxic stress and impaired neuronal homeostasis. (C) 2019 Elsevier Ltd. All rights reserved.

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.

RNA modifications are diverse post-transcriptional modifications that regulate RNA metabolism and gene expression. RNA modifications, and the writers, erasers, and readers that catalyze these modifications, serve as important signaling machineries in cellular stress responses and disease pathogenesis. In response to stress, RNA modifications are mobilized to activate or inhibit the signaling pathways that combat stresses, including oxidative stress, hypoxia, therapeutic stress, metabolic stress, heat shock, DNA damage, and ER stress. The role of RNA modifications in response to these cellular stressors is context- and cell-type-dependent. Due to their pervasive roles in cell biology, RNA modifications have been implicated in the pathogenesis of different diseases, including cancer, neurologic and developmental disorders and diseases, and metabolic diseases. In this review, we aim to summarize the roles of RNA modifications in molecular and cellular stress responses and diseases.

Centriolar satellites comprise cytoplasmic granules that are located around the centrosome. Their molecular identification was first reported more than a quarter of a century ago. These particles are not static in the cell but instead constantly move around the centrosome. Over the last decade, significant advances in their molecular compositions and biological functions have been achieved due to comprehensive proteomics and genomics, super-resolution microscopy analyses and elegant genetic manipulations. Centriolar satellites play pivotal roles in centrosome assembly and primary cilium formation through the delivery of centriolar/centrosomal components from the cytoplasm to the centrosome. Their importance is further underscored by the fact that mutations in genes encoding satellite components and regulators lead to various human disorders such as ciliopathies. Moreover, the most recent findings highlight dynamic structural remodelling in response to internal and external cues and unexpected positive feedback control that is exerted from the centrosome for centriolar satellite integrity.

Made available in DSpace on 2018-11-26T15:29:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universidade Estadual de Sao Paulo - UNESP (PROINTER - PROPe/PROPG) Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10 mu g/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies. (C) 2016 Elsevier B.V. All rights reserved. Univ Estadual Paulista, Araraquara Sch Dent, Araraquara, SP, Brazil Univ Ribeirao Preto, UNAERP, Ribeirao Preto, SP, Brazil Univ Estadual Paulista, Araraquara Sch Dent, Araraquara, SP, Brazil FAPESP: 2013/05879-0 FAPESP: PD: 2012/17947-8 CNPq: 303599/2014-6 Universidade Estadual de Sao Paulo - UNESP (PROINTER - PROPe/PROPG): 907 - PROINTER - PROPe/PROPG

In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information.

Contains fulltext : 168248.pdf (Publisher’s version ) (Open Access) Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses. 14 p.

Pyroptosis is one special type of lytic programmed cell death, featured in cell swelling, rupture, secretion of cell contents and remarkable proinflammation effect. In the process of pyroptosis, danger signalling and cellular events are detected by inflammasome, activating caspases and cleaving Gasdermin D (GSDMD), along with the secretion of IL-18 and IL-1β. Pyroptosis can be divided into canonical pathway and non-canonical pathway, and NLRP3 inflammasome is the most important initiator. Diabetic kidney disease (DKD) is one of the most serious microvascular complications in diabetes. Current evidence reported the stimulatory role of hyperglycaemia-induced cellular stress in renal cell pyroptosis, and different signalling pathways have been shown to regulate pyroptosis initiation. Additionally, the inflammation and cellular injury caused by pyroptosis are tightly implicated in DKD progression, aggravating renal fibrosis, glomerular sclerosis and tubular injury. Some registered hypoglycaemia agents exert suppressive activity in pyroptosis regulation pathway. Latest studies also reported some potential approaches to target the pyroptosis pathway, which effectively inhibits renal cell pyroptosis and alleviates DKD in in vivo or in vitro models. Therefore, comprehensively compiling the information associated with pyroptosis regulation in DKD is the main aim of this review, and we try to provide new insights for researchers to dig out more potential therapies of DKD.

The suborder Notothenioidae is comprised of Antarctic fishes, several of which have lost their ability to rapidly upregulate heat shock proteins in response to thermal stress, instead adopting a pattern of expression resembling constitutive genes. Given the cold-denaturing effect that sub-zero waters have on proteins, evolution in the Southern Ocean has likely selected for increased expression of molecular chaperones. These selective pressures may have also enabled retention of gene duplicates, bolstering quantitative output of cytosolic heat shock proteins (HSPs). Given that newly duplicated genes are under more relaxed selection, it is plausible that gene duplication enabled altered regulation of such highly conserved genes. To test for evidence of gene duplication, copy number of various isoforms within major heat shock gene families were characterized via qPCR and compared between the Antarctic notothen, Trematomus bernacchii, which lost the inducible heat shock response, and the non-Antarctic notothen, Notothenia angustata, which maintains an inducible heat shock response. The results indicate duplication of isoforms within the hsp70 and hsp40 super families have occurred in the genome of T. bernacchii. The findings suggest gene duplications may have been critical in maintaining protein folding efficiency in the sub-zero waters and provided an evolutionary mechanism of alternative regulation of these conserved gene families.

Objective The antioxidant activity and protective effect of a methanolic extract obtained from themarine Gram-negative bacterium Novosphingobium sp. PP1Y, isolated from the surface water of a polluted area in the harbour of Pozzuoli (Naples, Italy), was evaluated. Results The extract was tested in vitro on epithelial colorectal adenocarcinoma cells and in vivo on Caenorhabditis elegans. It showed strong protectiveactivity against oxidative stress, in both experimental systems, by preventing ROS accumulation. In the case of the cells, pre-treatment with methanolic extract was also able to maintain unaltered intracellular GSH levels and phosphorylation levels of mitogen-activated protein kinases p38. Instead, in the case of the worms, the extract was able to modulate the expression levels of stress response genes, by activating the transcription factor skn-1. Conclusions From a biotechnological and economical point of view, antioxidants from microorganisms are convenient as they provide a valid alternative to chemical synthesis and respond to the ever-growing market demand for natural antioxidants.

Cellular reprogramming and induced pluripotent stem cell (IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine. While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium- and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues (such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.

Macroautophagy (autophagy) is an evolutionarily conserved recycling and stress response mechanism. Active at basal levels in eukaryotes, autophagy is upregulated under stress providing cells with building blocks such as amino acids. A lysosome-integrated sensor system composed of RRAG GTPases and MTOR complex 1 (MTORC1) regulates lysosome biogenesis and autophagy in response to amino acid availability. Stress-mediated inhibition of MTORC1 results in the dephosphorylation and nuclear translocation of the TFE/MITF family of transcriptional factors, and triggers an autophagy- and lysosomal-related gene transcription program. The role of family members TFEB and TFE3 have been studied in detail, but the importance of MITF proteins in autophagy regulation is not clear so far. Here we introduce for the first time a specific role for MITF in autophagy control that involves upregulation of MIR211. We show that, under stress conditions including starvation and MTOR inhibition, a MITF-MIR211 axis constitutes a novel feed-forward loop that controls autophagic activity in cells. Direct targeting of the MTORC2 component RICTOR by MIR211 led to the inhibition of the MTORC1 pathway, further stimulating MITF translocation to the nucleus and completing an autophagy amplification loop. In line with a ubiquitous function, MITF and MIR211 were co-expressed in all tested cell lines and human tissues, and the effects on autophagy were observed in a cell-type independent manner. Thus, our study provides direct evidence that MITF has rate-limiting and specific functions in autophagy regulation. Collectively, the MITF-MIR211 axis constitutes a novel and universal autophagy amplification system that sustains autophagic activity under stress conditions. Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AKT1S1/PRAS40: AKT1 substrate 1; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; DEPTOR: DEP domain containing MTOR interacting protein; GABARAP: GABA type A receptor-associated protein; HIF1A: hypoxia inducible factor 1 subunit alpha; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPKAP1/SIN1: mitogen-activated protein kinase associated protein 1; MITF: melanogenesis associated transcription factor; MLST8: MTOR associated protein, LST8 homolog; MRE: miRNA response element; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; PRR5/Protor 1: proline rich 5; PRR5L/Protor 2: proline rich 5 like; RACK1: receptor for activated C kinase 1; RPTOR: regulatory associated protein of MTOR complex 1; RICTOR: RPTOR independent companion of MTOR complex 2; RPS6KB/p70S6K: ribosomal protein S6 kinase; RT-qPCR: quantitative reverse transcription-polymerase chain reaction; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TSC1/2: TSC complex subunit 1/2; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; VIM: vimentin; VPS11: VPS11, CORVET/HOPS core subunit; VPS18: VPS18, CORVET/HOPS core subunit; WIPI1: WD repeat domain, phosphoinositide interacting 1.