Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Extracellular Vesicles based STAT3 delivery as innovative therapeutic approach to restore STAT3 signaling deficiency.

Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations.

Essential oils and their nanoformulations for breast cancer therapy.

Breast Cancer (BC) is the most prevalent type of cancer in the world. Current treatments include surgery, radiation, and chemotherapy but often are associated with high toxicity to normal tissues, chemoresistance, and relapse. Thus, developing novel therapies which could combat these limitations is essential for effective treatment. In this context, phytochemicals are increasingly getting popular due to their safety profile, ability to efficiently target tumors, and circumvent limitations of existing treatments. Essential Oils (EOs) are mixtures of various phytochemicals which have shown potential anticancer activity in preclinical BC models. However, their clinical translation is limited by factors such as high volatility, low stability, and poor solubility. Nanotechnology has facilitated their encapsulation in a variety of nanostructures and proven to overcome these limitations. In this review, we have efficiently summarized the current knowledge on the anticancer effect of EOs and constituents in both in in vitro and in in vivo BC models. Further, we also provide a descriptive account on the potential of nanotechnology in enhancing the anti-BC activity of EOs and their constituents. The papers discussed in this review were selected using the keywords "antiproliferative Essential Oils in breast cancer," "anticancer activity of Essential Oil in breast cancer," and "cytotoxicity of Essential Oils in breast cancer" performed in PubMed and ScienceDirect databases.

Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus.

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.

Slow Evolution toward "Super-Aggregation" of the Oligomers Formed through the Swapping of RNase A N-Termini: A Wish for Amyloidosis?

Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.

Pinus mugo Essential Oil Impairs STAT3 Activation through Oxidative Stress and Induces Apoptosis in Prostate Cancer Cells.

Essential oils (EOs) and their components have been reported to possess anticancer properties and to increase the sensitivity of cancer cells to chemotherapy. The aim of this work was to select EOs able to downregulate STAT3 signaling using Western blot and RT-PCR analyses. The molecular mechanism of anti-STAT3 activity was evaluated through spectrophotometric and fluorometric analyses, and the biological effect of STAT3 inhibition was analyzed by flow cytometry and wound healing assay. Herein, Pinus mugo EO (PMEO) is identified as an inhibitor of constitutive STAT3 phosphorylation in human prostate cancer cells, DU145. The down-modulation of the STAT3 signaling cascade decreased the expression of anti-proliferative as well as anti-apoptotic genes and proteins, leading to the inhibition of cell migration and apoptotic cell death. PMEO treatment induced a rapid drop in glutathione (GSH) levels and an increase in reactive oxygen species (ROS) concentration, resulting in mild oxidative stress. Pretreatment of cells with N-acetyl-cysteine (NAC), a cell-permeable ROS scavenger, reverted the inhibitory action of PMEO on STAT3 phosphorylation. Moreover, combination therapy revealed that PMEO treatment displayed synergism with cisplatin in inducing the cytotoxic effect. Overall, our data highlight the importance of STAT3 signaling in PMEO cytotoxic activity, as well as the possibility of developing adjuvant therapy or sensitizing cancer cells to conventional chemotherapy.

[Emergency card, emergency medication, and information leaflet for the prevention and treatment of adrenal crisis (Addison crisis): an Austrian consensus document]. / Notfallausweis, Notfallmedikation und Informationsmaterial zur Prävention und Therapie der Nebennierenkrise (Addison-Krise): Ein österreichisches Konsensusdokument.

A central goal of the adrenal insufficiency management is the prevention of acute adrenal insufficiency (also known as adrenal crisis or Addison crisis). This consensus document was generated in order to achieve better implementation and harmonization of measures for the prevention and treatment of acute adrenal insufficiency in Austria. The following measures are generally recommended for all patients with adrenal insufficiency and are outlined in this manuscript: (1) Provision of a "steroid emergency card" and possibly also a medical alert bracelet or necklace (or similar identification). (2) Provision of a hydrocortisone injection kit (or alternative glucocorticoid preparations) for emergency use plus sufficient oral glucocorticoid doses for stress situations/illness. (3) Education of patients and relatives on glucocorticoid stress dosing and "sick day rules" as well as on self-injection of hydrocortisone. (4) Provision of a treatment guideline (information leaflet) for the prevention and therapy of the adrenal crisis, which should also be shown to healthcare staff if necessary. (5) Provision of an emergency phone number (contact details) of the responsible endocrine specialist team or other trained staff. (6) Reinforcement of patient education on a regular basis (preferably yearly). This consensus document also includes recommendations for glucocorticoid dosing in the perioperative setting as well as in various other stress situations.

Redox Sensitive Cysteine Residues as Crucial Regulators of Wild-Type and Mutant p53 Isoforms.

The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the TP53 gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role. This dual role of the wild-type protein and the mutated isoforms is also evident in the regulation of the redox state of the cell, with antioxidant and prooxidant functions, respectively. In this review, we introduce a new concept of the p53 protein by discussing its sensitivity to the cellular redox state. In particular, we focus on the discussion of structural and functional changes following post-translational modifications of redox-sensitive cysteine residues, which are also responsible for interacting with zinc ions for proper structural folding. We will also discuss therapeutic opportunities using small molecules targeting cysteines capable of modifying the structure and function of the p53 mutant isoforms in view of possible anticancer therapies for patients possessing the mutation in the TP53 gene.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

The anti-STAT1 polyphenol myricetin inhibits M1 microglia activation and counteracts neuronal death.

Microglia activation toward M1 pro-inflammatory phenotype represents one of the earliest events of neurological disorders. Therefore, reducing microglia activation should inhibit neuroinflammation, thereby delaying the progression of neurodegeneration. Recently, we pointed out the role of STAT1 signaling in hypoxia-induced M1 activation and proposed STAT1 as a suitable molecular target for the prevention and treatment of neurodegeneration. Myricetin (MYR) is a natural flavonoid that exhibits a specific anti-STAT1 activity correlated with its direct interaction with STAT1 protein itself. Herein, we investigated the anti-inflammatory effect of MYR and its ability to protect neurons from death in an in vitro model of neurotoxicity using the neuroblast-like SH-SY5Y cells that were exposed to conditioned media from hypoxia-activated microglia BV2 cells. We demonstrate that MYR pretreatment is able to switch off hypoxia-induced M1 microglia polarization through the inhibition of STAT1 signaling. The analysis of the molecular mechanism suggests that the direct interaction of MYR with STAT1 impairs its S-glutathionylation and phosphorylation. Moreover, treatment of SH-SY5Y cells with conditioned medium from hypoxia-activated microglia pretreated with MYR produced a significant reduction in neuronal viability. Our data indicate that MYR may represent a promising candidate for prevention and treatment of neuroinflammation in neurodegenerative disorders.

Redox Regulation of STAT1 and STAT3 Signaling.

STAT1 and STAT3 are nuclear transcription factors that regulate genes involved in cell cycle, cell survival and immune response. The cross-talk between these signaling pathways determines how cells integrate the environmental signals received ultimately translating them in transcriptional regulation of specific sets of genes. Despite being activated downstream of common cytokine and growth factors, STAT1 and STAT3 play essentially antagonistic roles and the disruption of their balance directs cells from survival to apoptotic cell death or from inflammatory to anti-inflammatory responses. Different mechanisms are proposed to explain this yin-yang relationship. Considering the redox aspect of STATs proteins, this review attempts to summarize the current knowledge of redox regulation of STAT1 and STAT3 signaling focusing the attention on the post-translational modifications that affect their activity.

Rehabilitation and Biomarkers of Stroke Recovery: Study Protocol for a Randomized Controlled Trial.

Background: Stroke is a leading cause of disability. Nonetheless, the care pathway for stroke rehabilitation takes partially into account the needs of chronic patients. This is due in part to the lack of evidence about the mechanisms of recovery after stroke, together with the poor knowledge of related and influencing factors. Here we report on the study protocol "Rehabilitation and Biomarkers of Stroke Recovery," which consists of 7 work-packages and mainly aim to investigate the effects of long-term neurorehabilitation on stroke patients and to define a related profile of (clinical-biological, imaging, neurophysiological, and genetic-molecular) biomarkers of long-term recovery after stroke. The work-package 1 will represent the main part of this protocol and aims to compare the long-term effects of intensive self-rehabilitation vs. usual (rehabilitation) care for stroke. Methods: We planned to include a total of 134 adult subacute stroke patients (no more than 3 months since onset) suffering from multidomain disability as a consequence of first-ever unilateral ischemic stroke. Eligible participants will be randomly assigned to one of the following groups: intensive self-rehabilitation (based on the principles of "Guided Self-Rehabilitation Contract") vs. usual care (routine practice). Treatment will last 1 year, and patients will be evaluated every 3 months according to their clinical presentation. The following outcomes will be considered in the main work-package: Fugl-Meyer assessment, Cognitive Oxford Screen Barthel Index, structural and functional neuroimaging, cortical excitability, and motor and somatosensory evoked potentials. Discussion: This trial will deal with the effects of an intensive self-management rehabilitation protocol and a related set of biomarkers. It will also investigate the role of training intensity on long-term recovery after stroke. In addition, it will define a set of biomarkers related to post-stroke recovery and neurorehabilitation outcome in order to detect patients with greater potential and define long-term individualized rehabilitation programs. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT04323501.

Immunoprecipitation methods to identify S-glutathionylation in target proteins.

S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function. Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins. Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein. •S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.•Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.•The setups of both methods guarantee high reproducibility.

Natural Sesquiterpene Lactones Enhance Chemosensitivity of Tumor Cells through Redox Regulation of STAT3 Signaling.

STAT3 is a nuclear transcription factor that regulates genes involved in cell cycle, cell survival, and immune response. Although STAT3 activation drives cells to physiological response, its deregulation is often associated with the development and progression of many solid and hematological tumors as well as with drug resistance. STAT3 is a redox-sensitive protein, and its activation state is related to intracellular GSH levels. Under oxidative conditions, STAT3 activity is regulated by S-glutathionylation, a reversible posttranslational modification of cysteine residues. Compounds able to suppress STAT3 activation and, on the other hand, to modulate intracellular redox homeostasis may potentially improve cancer treatment outcome. Nowadays, about 35% of commercial drugs are natural compounds that derive from plant extracts used in phytotherapy and traditional medicine. Sesquiterpene lactones are an interesting chemical group of plant-derived compounds often employed in traditional medicine against inflammation and cancer. This review focuses on sesquiterpene lactones able to downmodulate STAT3 signaling leading to an antitumor effect and correlates the anti-STAT3 activity with their ability to decrease GSH levels in cancer cells. These properties make them lead compounds for the development of a new therapeutic strategy for cancer treatment.

Tumor Dormancy and Interplay with Hypoxic Tumor Microenvironment.

The tumor microenvironment is a key factor in disease progression, local resistance, immune-escaping, and metastasis. The rapid proliferation of tumor cells and the aberrant structure of the blood vessels within tumors result in a marked heterogeneity in the perfusion of the tumor tissue with regions of hypoxia. Although most of the tumor cells die in these hypoxic conditions, a part of them can adapt and survive for many days or months in a dormant state. Dormant tumor cells are characterized by cell cycle arrest in G0/G1 phase as well as a low metabolism, and are refractive to common chemotherapy, giving rise to metastasis. Despite these features, the cells retain their ability to proliferate when conditions improve. An understanding of the regulatory machinery of tumor dormancy is essential for identifying early cancer biomarkers and could provide a rationale for the development of novel agents to target dormant tumor cell populations. In this review, we examine the current knowledge of the mechanisms allowing tumor dormancy and discuss the crucial role of the hypoxic microenvironment in this process.

Biopsychosocial model of resilience in young adults with multiple sclerosis (BPS-ARMS): an observational study protocol exploring psychological reactions early after diagnosis.

INTRODUCTION: Multiple sclerosis (MS), the most common neurological disease causing disability in young adults, is widely recognised as a major stress factor. Studies have shown that the first years after the diagnosis are distressing in terms of adjustment to the disease and that MS negatively affects patients' psychological well-being, quality of life (QoL) and social functioning. However, the links between disease-specific variables at diagnosis, resilience and psychological adjustment of patients with MS remain largely unexplored, especially in adolescents and young adults. This observational study aims to fill the gap of knowledge on biopsychosocial characteristics and resilience of young adults with MS to evaluate the relationship among these variables and to develop a biopsychosocial model of resilience. METHODS AND ANALYSIS: Biological and clinical characteristics of young adults newly diagnosed with MS will be investigated by collecting clinical information, performing neurological examinations, MRI and analysing cerebrospinal fluid and blood biomarkers (eg, measures of inflammation), body composition, gut microbiota and movement/perceptual markers. Psychosocial characteristics (eg, psychological distress, coping strategies), QoL, psychological well-being and resilience will be assessed by self-report questionnaires. Comparative statistics (ie, analysis of variance or unpaired samples t-test, correlation and regression analyses) will be applied to evaluate the relationship among biological, psychological and social factors. The results are expected to allow a comprehensive understanding of the determinants of resilience in young patients with MS and to inform resilience interventions, tailored to young patients' specific needs, aiming to reduce the risk of maladaptive reactions to the disease and to improve psychological well-being and QoL. ETHICS AND DISSEMINATION: The study has been approved by the Verona University Hospital Ethics Committee (approval number: 2029CESC). The findings will be disseminated through scientific publications in peer-reviewed journals, conference presentations, social media and specific websites. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT03825055).

STAT1 drives M1 microglia activation and neuroinflammation under hypoxia.

Microglia are resident immune cells that act as the first active defence in the central nervous system. These cells constantly monitor the tissue microenvironment and rapidly react in response to hypoxia, infection and injuries. Hypoxia in the brain has been detected in several neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Hypoxic conditions activate microglia cells towards M1 phenotype resulting in oxidative stress and the release of pro-inflammatory cytokines. Recently, we have demonstrated that oxidative stress induces S-glutathionylation of the STAT1 and hyper-activates its signaling in microglia BV2 cells pointing out the importance of this transcription factor in neuroinflammation. In this paper we analyse the cellular mechanisms that drive M1 microglia activation in BV2 cells in response to hypoxia correlating it to STAT1 activation. The analysis of the molecular mechanism of STAT1 signaling reveals that hypoxia generates oxidative stress and induces both phosphorylation and S-glutathionylation of STAT1 that are responsible of its aberrant activation. The silencing of STAT1 protein expression counteracts hypoxia-M1 microglia phenotype suggesting the strong link between hypoxia-STAT1 and STAT1-microglia activation.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2-· production in cancer cells.

BACKGROUND: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy. METHODS: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection. RESULTS: We demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2-· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-L-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs. CONCLUSIONS: The inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.

S-glutathionylation exerts opposing roles in the regulation of STAT1 and STAT3 signaling in reactive microglia.

STAT1 and STAT3 are two transcription factors involved in a lot of cellular functions such as immune response, proliferation, apoptosis, and cell survival. A number of literature evidences described a yin-yang relationship between activation of STAT1 and STAT3 in neurodegenerative disorders where STAT1 exerts a pro-apoptotic effect whereas STAT3 shows neuroprotective properties through the inhibition of apoptosis. Although the role of oxidative-stress in the pathogenesis of neurodegeneration is clearly described, its influence in the regulation of these pathways is poorly understood. Herein, we demonstrate that H2O2 rapidly induces phosphorylation of STAT1 whereas it is not able to influence phosphorylation of STAT3 in mouse microglia BV2 cells. The analysis of the molecular mechanism of STATs signaling reveals that H2O2 induces S-glutathionylation of both STAT1 and STAT3. The same post-translational event exerts an opposing role in the regulation of STAT1 and STAT3 signaling. These data not only confirm redox sensibility of STAT3 signaling but also reveal for the first time that STAT1 is susceptible to redox regulation. A deep study of the molecular mechanism of STAT1 redox regulation, identifies Cys324 and Cys492 as the main targets of S-glutathionylation and confirms that S-glutathionylation does not impair JAK2 mediated STAT1 tyrosine phosphorylation. These results demonstrate that both phosphorylation and glutathionylation contribute to activation of STAT1 during oxidative stress and underline that the same post-translation event exerts an opposing role in the regulation of STAT1 and STAT3 signaling in microglia cells.

Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in cancer cells.

Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory 'pancreatic-type' RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.