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Reactive oxygen species (ROS) are currently recognized as a key driver of several physiological processes. Increasing evidence indicates that ROS levels can affect myogenic differentiation, but the molecular mechanisms still need to be elucidated. Protein kinase C (PKC) epsilon (PKCe) promotes muscle stem cell differentiation and regeneration of skeletal muscle after injury. PKCs play a tissue-specific role in redox biology, with specific isoforms being both a target of ROS and an up-stream regulator of ROS production. Therefore, we hypothesized that PKCe represents a molecular link between redox homeostasis and myogenic differentiation. We used an in vitro model of a mouse myoblast cell line (C2C12) to study the PKC–redox axis. We demonstrated that the transition from a myoblast to myotube is typified by increased PKCe protein content and decreased ROS. Intriguingly, the expression of the antioxidant enzyme superoxide dismutase 2 (SOD2) is significantly higher in the late phases of myogenic differentiation, mimicking PKCe protein content. Furthermore, we demonstrated that PKCe inhibition increases ROS and reduces SOD2 protein content while SOD2 silencing did not affect PKCe protein content, suggesting that the kinase could be an up-stream regulator of SOD2. To support this hypothesis, we found that in C2C12 cells, PKCe interacts with Nrf2, whose activation induces SOD2 transcription. Overall, our results indicate that PKCe is capable of activating the antioxidant signaling preventing ROS accumulation in a myotube, eventually promoting myogenic differentiation.

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein kinase C epsilon (PKCϵ) has recently emerged as a regulator of several cell-cycle processes associated with the resolution of mitotic catenation during the metaphase–anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCϵ in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCϵ controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCϵ dependency of mitotic spindle organization is correlated with the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a characteristic of many transformed cells. Thus, PKCϵ appears to become specifically engaged in a programme of controls that are required to support cell-cycle progression in transformed cells, advocating for PKCϵ as a potential cancer therapeutic target.Implications: The close relationship between PKCϵ dependency for mitotic spindle organization and the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a hallmark of transformed cells, strongly suggests PKCϵ as a therapeutic target in cancer. Mol Cancer Res; 16(1); 3–15. ©2017 AACR.

This figure show that Ciliobrevin treatment results in mitotic spindle defects, delay of centrosome movement and bending phenotype.

Here are described the Supplementary Figures from Figure S1 to Figure S4.

Supplemental Figure Legends

Supplemental Methods

Supplemental Figure 7. (A-D) OCI-AML3 cells were transduced with lentiviruses expressing CTRL shRNA or SOD2 shRNA and analyzed for: (A) Western blot analysis 4 days after transduction with the indicated antibodies. (B) MitoSOX and Annexin V staining by flow cytometry, 4 days after transduction. The bar graph shows the MitoSOX median fluorescence intensity (MFI) of live (Annexin V-) cells and data is expressed as mean {plus minus} SD of three independent experiments. (C) Cell growth every 2 days by MTS analysis. Data represents the mean {plus minus} SD of three independent replicates. (D) Annexin V staining 5 days after transduction. Data represents the mean {plus minus} SD of three independent replicates. (E) Bar graph representing the number of colonies formed by mouse MLL-AF9 leukemia cells transduced with CTRL shRNA, Sod2 shRNA_1 and Sod2 shRNA_2 in methylcellulose culture. Data is expressed as mean {plus minus} SD of three independent replicates. (*p{less than or equal to}0.05, **p{less than or equal to}0.01; ****p

Supplemental Figure 6. (A & B) OCI-AML3 cells were transduced with CTRL shRNA, PKCε shRNA_1 or PKCε shRNA_2 and 24 hours later cells from each condition were treated with 100µM NAC (A) or 50µM glutathione (B), every 12 hours until the end of the experiment. Four days after treatment, cells from each condition were assessed for Annexin V staining by flow cytometry (****p

Supplemental Table 1. Clinical characteristics and mutation status of primary AML samples.

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown.Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCϵ) in intracellular redox biology, cell survival and disease progression.Results: We show that RNA interference-mediated inhibition of PKCϵ significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCϵ inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCϵ inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo. Furthermore, we report that PKCϵ over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCϵ may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport.Conclusions: This study uncovers a previously unrecognized role for PKCϵ in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608–18. ©2017 AACR.

T cell-mediated adaptive immunity is designed to respond to non-self antigens and pathogens through the activation and proliferation of various T cell populations. T helper 1 (Th1), Th2, Th17 and Treg cells finely orchestrate cellular responses through a plethora of paracrine and autocrine stimuli that include cytokines, autacoids, and hormones. Hydrogen sulfide (H2S) is one of these mediators able to induce/inhibit immunological responses, playing a role in inflammatory and autoimmune diseases, neurological disorders, asthma, acute pancreatitis, and sepsis. Both endogenous and exogenous H2S modulate numerous important cell signaling pathways. In monocytes, polymorphonuclear, and T cells H2S impacts on activation, survival, proliferation, polarization, adhesion pathways, and modulates cytokine production and sensitivity to chemokines. Here, we offer a comprehensive review on the role of H2S as a natural buffer able to maintain over time a functional balance between Th1, Th2, Th17 and Treg immunological responses.

Clinical course and outcome of viral infections mostly depends on the adequate orchestration of both innate and adaptive immune response to the viral noxa. The loss of this balance emerged to be critical during SARS-CoV-2 infection, in which an unleashed cytokine response (the so called “cytokine storm”) has been implicated as the main cause of morbidity and mortality in COVID-19 cases [[1]Liu J Li S Liu J et al.Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.EBioMedicine. 2020; 55102763Summary Full Text Full Text PDF PubMed Scopus (516) Google Scholar]. Therefore, disrupting the escalation of molecular events that lead to severe COVID-19 is an urgent - and still unmet- medical need.

Sulphurous thermal water inhalations have been traditionally used in the treatment of airway diseases. In vivo and in vitro studies reported that they ameliorate mucus rheology, mucociliary clearance and reduce inflammation. Cigarette smoking induces an inflammatory damage, with consequent remodeling of respiratory airways, which in turn affect pulmonary functions. Despite the anti-inflammatory effects of H2S are clinically documented in several airway inflammatory diseases, data on the effects of sulphurous thermal water treatment on pulmonary function and biomarkers of airways inflammation in smokers are still scant. Therefore, we investigated whether a conventional cycle of sulphurous thermal water inhalation produced changes in markers of respiratory inflammation and function. A cohort of 504 heavy current and former smokers underwent 10-day cycles of sulphurous thermal water inhalation. Pulmonary function and metabolic analyses on exhaled breath condensate were then performed at day 0 and after the 10-day treatment. Spirometric data did not change after spa therapy, while exhaled breath condensate analysis revealed that a single 10-day cycle of sulphurous water inhalation was sufficient to induce a statistically significant increase of citrulline levels along with a decrease in ornithine levels, thus shifting arginine metabolism towards a reduced nitric oxide production, i.e. an anti-inflammatory profile. Overall, sulphurous thermal water inhalation impacts on arginine catatabolic intermediates of airways cells, shifting their metabolic balance towards a reduction of the inflammatory activity, with potential benefits for smokers.

A rapid differential diagnosis of the clinical conditions underlying chest pain is a relevant clinical issue. Specifically, a fast rule-in or -out of acute myocardial infarction (AMI) is mandatory to improve diagnostic outcome and cost-effectiveness of patient management. We demonstrated that Protein Kinase C (PKC) epsilon is selectively expressed by platelets from AMI patients, accounting for increased platelet activation. Thus, we hypothesized that PKCepsilon-expressing platelets may represent a pathophysiological marker of AMI that could be utilized in combination with troponin-I, the conventional marker of cardiac injury, to add diagnostic information in chest pain workup. In 94 chest pain patients consecutively admitted to Parma University Hospital, we tested the diagnostic performance of flow-cytometric detection of PKCepsilon expressing platelets in discriminating AMI vs. non-AMI conditions. We demonstrated that PKCepsilon-expressing platelets were significantly higher in patients with AMI. Flow cytometry detection of PKCepsilon-expressing platelets showed high sensitivity and specificity (87.5% and 84.4%, respectively) and good diagnostic accuracy (AUC: 0.875). The combination of PKCepsilon expressing platelets and cardiac troponin clearly discriminates patients with 100% and 0% of probability to be affected by AMI. Overall, we highlighted a dual marker strategy potentially useful for a rapid rule-in or -out of myocardial infarction in chest pain patients.

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2—one of the most potent immunomodulatory chemokines—to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that (i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes, (ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor), (iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation, (iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.

BACKGROUND: After ruxolitinib discontinuation, the outcome of patients with myelofibrosis (MF) is poor with scarce therapeutic possibilities. METHODS: The authors performed a subanalysis of an observational, retrospective study (RUX-MF) that included 703 MF patients treated with ruxolitinib to investigate 1) the frequency and reasons for ruxolitinib rechallenge, 2) its therapeutic effects, and 3) its impact on overall survival. RESULTS: A total of 219 patients (31.2%) discontinued ruxolitinib for ≥14 days and survived for ≥30 days. In 60 patients (27.4%), ruxolitinib was rechallenged for ≥14 days (RUX-again patients), whereas 159 patients (72.6%) discontinued it permanently (RUX-stop patients). The baseline characteristics of the 2 cohorts were comparable, but discontinuation due to a lack/loss of spleen response was lower in RUX-again patients (P =.004). In comparison with the disease status at the first ruxolitinib stop, at its restart, there was a significant increase in patients with large splenomegaly (P 10 mg twice daily predicted better spleen (P =.05) and symptom improvements (P =.02), the reasons for/duration of ruxolitinib discontinuation and the use of other therapies before rechallenge were not associated with rechallenge efficacy. At 1 and 2 years, 33.3% and 48.3% of RUX-again patients, respectively, had permanently discontinued ruxolitinib. The median overall survival was 27.9 months, and it was significantly longer for RUX-again patients (P =.004). CONCLUSIONS: Ruxolitinib rechallenge was mainly used in intolerant patients; there were clinical improvements and a possible survival advantage in many cases, but there was a substantial rate of permanent discontinuation. Ruxolitinib rechallenge should be balanced against newer therapeutic possibilities.

Reactive Oxygen Species (ROS) are molecules naturally produced by cells. If their levels are too high, the cellular antioxidant machinery intervenes to bring back their quantity to physiological conditions. Since aging often induces malfunctioning in this machinery, ROS are considered an effective cause of age-associated diseases. Exercise stimulates ROS production on one side, and the antioxidant systems on the other side. The effects of exercise on oxidative stress markers have been shown in blood, vascular tissue, brain, cardiac and skeletal muscle, both in young and aged people. However, the intensity and volume of exercise and the individual subject characteristics are important to envisage future strategies to adequately personalize the balance of the oxidant/antioxidant environment. Here, we reviewed the literature that deals with the effects of physical activity on redox balance in young and aged people, with insights into the molecular mechanisms involved. Although many molecular pathways are involved, we are still far from a comprehensive view of the mechanisms that stand behind the effects of physical activity during aging. Although we believe that future precision medicine will be able to transform exercise administration from wellness to targeted prevention, as yet we admit that the topic is still in its infancy.

The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na+/H+ exchanger (NHE) was also explored., info:eu-repo/semantics/published

Natural killer (NK) cells are pivotal effectors of the innate immunity protecting an individual from microbes. They are the first line of defense against invading viruses, given their substantial ability to directly target infected cells without the need for specific antigen presentation. By establishing cellular networks with a variety of cell types such as dendritic cells, NK cells can also amplify and modulate antiviral adaptive immune responses. In this review, we will examine the role of NK cells in SARS-COV2 infections causing the ongoing COVID19 pandemic, keeping in mind the controversial role of NK cells specifically in viral respiratory infections and in inflammatory-driven lung damage. We discuss lessons learnt from previous coronavirus outbreaks in humans (caused by SARS-CoV-1 and MERS-COV).

PKCe is implicated in T cell activation and proliferation and is overexpressed in CD4+ -T cells from patients with autoimmune Hashimoto's thyroiditis. Although this might induce the suspicion that PKCe takes part in autoimmunity, its role in the molecular pathophysiology of immune-mediated disorders is still largely unknown. We studied PKCe expression in circulating CD4+ -T cells from patients with psoriasis, a skin disorder characterized by an increased amount of Th17 cells, a CD4+ subset that is critical in the development of autoimmunity. Although the mechanisms that underlie Th17 differentiation in humans are still unclear, we here show that: (i) PKCe is overexpressed in CD4+ -T cells from psoriatic patients, and its expression positively correlates with the severity of the disease, being reduced by effective phototherapy; (ii) PKCe interacts with Stat3 during Th17 differentiation and its overexpression results in an enhanced expression of Stat3 and pStat3(Ser727); iii) conversely, when PKCe is forcibly downregulated, CD4+ -T cells show lower levels of pStat3(Ser727) expression and defective in vitro expansion into the Th17-lineage. These data provide a novel insight into the molecular mechanisms of Th17 cell polarization that is known to play a crucial role in autoimmunity, pinpointing PKCe as a potential target in Th17-mediated diseases.

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein kinase C epsilon (PKCϵ) has recently emerged as a regulator of several cell-cycle processes associated with the resolution of mitotic catenation during the metaphase–anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCϵ in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCϵ controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCϵ dependency of mitotic spindle organization is correlated with the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a characteristic of many transformed cells. Thus, PKCϵ appears to become specifically engaged in a programme of controls that are required to support cell-cycle progression in transformed cells, advocating for PKCϵ as a potential cancer therapeutic target. Implications: The close relationship between PKCϵ dependency for mitotic spindle organization and the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a hallmark of transformed cells, strongly suggests PKCϵ as a therapeutic target in cancer. Mol Cancer Res; 16(1); 3–15. ©2017 AACR.

The COVID-19 pandemic has now affected around 190 million people worldwide, accounting for more than 4 million confirmed deaths. Besides ongoing global vaccination, finding protective and therapeutic strategies is an urgent clinical need. SARS-CoV-2 mostly infects the host organism via the respiratory system, requiring angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enter target cells. Therefore, these surface proteins are considered potential druggable targets. Hydrogen sulfide (H2S) is a gasotransmitter produced by several cell types and is also part of natural compounds, such as sulfurous waters that are often inhaled as low-intensity therapy and prevention in different respiratory conditions. H2S is a potent biological mediator, with anti-oxidant, anti-inflammatory, and, as more recently shown, also anti-viral activities. Considering that respiratory epithelial cells can be directly exposed to H2S by inhalation, here we tested the in vitro effects of H2S-donors on TMPRSS2 and ACE2 expression in human upper and lower airway epithelial cells. We showed that H2S significantly reduces the expression of TMPRSS2 without modifying ACE2 expression both in respiratory cell lines and primary human upper and lower airway epithelial cells. Results suggest that inhalational exposure of respiratory epithelial cells to natural H2S sources may hinder SARS-CoV-2 entry into airway epithelial cells and, consequently, potentially prevent the virus from spreading into the lower respiratory tract and the lung.

Introduction: The outcome of patients (pts) with myelofibrosis (MF) who discontinue ruxolitinib (RUX) is poor with scarce therapeutic possibilities (Palandri et al, 2020). However, some evidences suggest that pts may respond to a rechallenge of RUX after drug stop (Gerds et al, 2018). Aims: To investigate in a real-world context: 1) frequency and reasons for rechallenge; 2) therapeutic effects of rechallenge; 3) impact of rechallenge on overall survival (OS) Methods: After IRB approval, a clinical database was created in 20 European Hematology Centers including now retrospective data of 703 MF pts who started RUX from Jan 2011 to Nov 2019. Only chronic phase (CP) pts who stopped RUX for ≥14 days and survived ≥30 days after discontinuation were included. A specific survey collected clinical/laboratory data at RUX stop and at rechallenge, reasons for discontinuation and treatments before rechallenge. OS was estimated from the date of the first/only RUX discontinuation to last contact (log-rank test). Results: A total of 219 CP pts was evaluable for this study. In 60 (27.4%) pts, RUX was re-challenged for ≥14 days after the first discontinuation (RUX-again), while 159 (72.6%) pts discontinued RUX permanently (RUX-stop). The median time from RUX start to stop was of 16.5 and 12.3 mos for RUX-again and RUX-stop pts, respectively (p=0.41). At RUX start, characteristics of RUX-again were: median age 67y (24-88); males 61.7%; PMF 53.3%; median Hb 10.2 g/dl; median PLT/WBC: 249/12.6 x109/l; median RUX starting dose: 15mg BID. Baseline characteristics of RUX-again and RUX-stop pts were comparable. In the 60 RUX-again pts, reasons for discontinuation included loss of/inadequate response (18 pts, 30%) and toxicity (42 pts, 70%). Toxicity included G3-4 thrombocytopenia (38.1%), anemia (26.2%), infections (21.4%), other (14.3%). Conversely, RUX-stop pts discontinued RUX mainly due to loss of/inadequate response (75 pts, 47.2%), while intolerable toxicity occurred in 69 pts (43.4%) (p=0.004) and other causes in 9.4%. At first RUX discontinuation, 35.7% of RUX-again pts presented with large (>10 cm) splenomegaly; median Total Symptoms Score (TSS) was 10 (TSS>20 in 30.4% of pts). The median duration of temporary RUX discontinuation was 2 mos (range 0.5-71.1). During RUX stop, 65% of RUX-again pts did not receive any therapy, 15% received only palliation (steroids, hydroxyurea), while 11.7% switched to investigational agents, 3.3% underwent splenectomy and 5% allogeneic transplantation. Compared to disease status at first RUX stop, at RUX restart there was a significant increase of pts with large splenomegaly and high TSS, while the PLT count was higher and RUX dose significantly lower (Table 1). The median duration of RUX rechallenge was 7.5 mos (0.5-72.7). During the rechallenge, 44.6% and 48.3% pts improved spleen and symptoms, and there was a significant increase in pts with TSS reduction (p=0.01); 8 pts (13.3%) continued RUX with stable/worsening spleen size and improvement in TSS. Conversely, 26.8% and 20% of pts had increase in spleen size and in symptoms, respectively. While Hb levels remained stable, PLT count significantly decreased during rechallenge (p The reasons for temporary discontinuation had no impact on the reduction of spleen/symptoms during rechallenge and on OS. However, comparing RUX-again and RUX-stop pts, RUX-again pts showed a better OS, with a median survival of 41.1 mos and 23.7, respectively in the 2 cohorts (Fig. 1). Conclusions: This real-world study highlights that RUX rechallenge is quite common in CP-MF pts, involving almost 30% of treated pts, particularly when the discontinuation is due to toxicity. The temporary discontinuation, while improving PLT count, generally caused a significant increase in disease burden. After rechallenge, almost 50% of pts achieved clinical responses regardless of reason of first discontinuation. This residual disease control activity, that correlated with improved OS, should be weighed up also given the new therapeutic possibilities available in these pts. Disclosures Palandri: Novartis: Consultancy, Honoraria. Breccia:Abbvie: Consultancy; Bristol-Myers Squibb/Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Benevolo:Amgen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Cavazzini:Incyte: Honoraria; Pfize: Honoraria; Novartis: Honoraria. Crugnola:Janssen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Celgene: Honoraria. Heidel:CTI: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Research Funding. Pane:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Other: travel expenses, Speakers Bureau; Daiichi Sankyo: Consultancy, Other: Travel Expenses; Novartis pharma SAS: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Other: Travel Expenses; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Other: Travel Expenses, Speakers Bureau; AbbVie: Consultancy, Other: Travel Expenses, Speakers Bureau. Cuneo:janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Krampera:Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Semenzato:Takeda: Honoraria; Roche: Honoraria; Abbvie: Honoraria. Lemoli:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BerGenBio ASA: Research Funding. Cavo:Jannsen, BMS, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, AbbVie, Karyopharm, Adaptive: Consultancy, Honoraria. Palumbo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background: The bench press exercise (BP) is commonly practiced in both recreational and professional training. The weight is lowered from a position where the elbows are at a 90&deg, angle at the start and &lt, 90&deg, at the end of eccentric phase, and then returned to the elbows extended position. In order to focus the exercise more on the triceps brachii (TB) rather than the pectoralis major (PM), the inter-handle distance (IHD) is decreased diminishing the involvement of the PM in favor of the TB. Purpose: To improve performance of the exercise by reducing force dissociation and transmitting 100% of the external load to the muscle tissue we propose a prototype of the barbell with a bar on which two sleeves are capable of sliding. The dynamic modifications of the IHD keep the elbow flexion angle constant at 90&deg, Results: Analysis of the inter-handle distance (IHD) signals of the upper body muscles showed a marked increase in muscle activity using the experimental barbell for the PM (19.5%) and for the biceps brachii (173%). Conclusions: The experimental barbell increased the muscle activity typical of the bench press exercise, obtaining the same training induction with a lower load and consequently preventing articular stress.

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

Preserving the hydration status means to balance daily fluids and salt losses with gains, where the losses depend on several physiological and environmental factors. Especially for athletes, these losses could be relevant and negatively influence the performance: therefore, their hydro-saline status must be preserved with personalized pre- and rehydration plans all along the performance period. Scientific literature in this field is mainly dedicated to artificial sport drinks. Different territories in most world areas are rich in drinking natural mineral spring waters with saline compositions that reflect their geological origin and that are used for human health (often under medical prescription). However, scarce scientific attention has been dedicated to the use of these waters for athletes. We therefore reviewed the existing literature from the innovative viewpoint of matching spring water mineral compositions with different athletic performances and their hydro-saline requirements.

Summary Objective Osteoarthritis (OA) is a common and highly debilitating degenerative disease whose complex pathogenesis and the multiplicity of the molecular processes involved, hinder its complete understanding. Protein Kinase C (PKC) novel isozyme PKCe recently proved to be an interesting molecule for further investigations as it can represent an intriguing, new actor in the acquisition of a OA phenotype by the chondrocyte. Design PKCe was modulated in primary chondrocytes from human OA patient knee cartilage samples by means of short hairpin RNA (ShRNA) and the expression of cartilage specific markers observed at mRNA and protein level. The involvement of Histone deacetylases (HDACs) signaling pathway was also investigated through the use of specific inhibitors MS-275 and Inhibitor VIII. Results PKCe loss induces up-regulation of Runt-domain transcription factor (RUNX2), Metalloproteinase 13 (MMP13) and Collagen X (COL10) as well as an enhanced calcium deposition in OA chondrocyte cultures. In parallel, PKCe knock-down also leads to SOX9 and Collagen II (COL2) down-modulation and to a lower deposition of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). This novel regulatory role of PKCe over cartilage hypertrophic phenotype is exerted via an HDAC-mediated pathway, as HDAC2 and HDAC4 expression is modulated by PKCe. HDAC2 and HDAC4, in turn, are at least in part responsible for the modulation of the master transcription factors RUNX2 and SOX9, key regulators of chondrocyte phenotype. Conclusions PKCe prevents the phenotypic progression of the OA chondrocyte, acting on cartilage specific markers through the modulation of the transcription factors SOX9 and RUNX2. The loss of PKCe enhances, in fact, the OA hypertrophic phenotype, with clear implications in the pathophysiology of the disease.

Reactive oxygen species (ROS) and mitochondria play a pivotal role in regulating platelet functions. Platelet activation determines a drastic change in redox balance and in platelet metabolism. Indeed, several signaling pathways have been demonstrated to induce ROS production by NAPDH oxidase (NOX) and mitochondria, upon platelet activation. Platelet-derived ROS, in turn, boost further ROS production and consequent platelet activation, adhesion and recruitment in an auto-amplifying loop. This vicious circle results in a platelet procoagulant phenotype and apoptosis, both accounting for the high thrombotic risk in oxidative stress-related diseases. This review sought to elucidate molecular mechanisms underlying ROS production upon platelet activation and the effects of an altered redox balance on platelet function, focusing on the main advances that have been made in platelet redox biology. Furthermore, given the increasing interest in this field, we also describe the up-to-date methods for detecting platelets, ROS and the platelet bioenergetic profile, which have been proposed as potential disease biomarkers.

Rationale Vessel formation is a crucial event in tissue repair after injury. Thus, one assumption of innovative therapeutic approaches is the understanding of its molecular mechanisms. Notwithstanding our knowledge of the role of Protein Kinase C epsilon (PKCe) in cardio-protection and vascular restenosis, its role in vessel progenitor differentiation remains elusive. Objective Given the availability of PKCe pharmacological modulators already tested in clinical trials, the specific aim of this study is to unravel the role of PKCe in vessel progenitor differentiation, with implications in vascular pathology and vasculogenesis. Methods and results Mouse Peri-Vascular Adipose Tissue (PVAT) was used as source of mesenchymal vessel progenitors. VEGF-induced differentiation of PVAT cells down-regulates both PKCe and p-PAK1 protein expression levels. PKCe overexpression and activation: i) reduced the expression levels of SMA and PECAM in endothelial differentiation of PVAT cells; ii) completely abrogated tubules formation in collagen gel assays; iii) increased the expression of p-PAK1. Conclusion PKCe negatively interferes with vessel progenitor differentiation via interaction with PAK-1.

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown. Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCϵ) in intracellular redox biology, cell survival and disease progression. Results: We show that RNA interference-mediated inhibition of PKCϵ significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCϵ inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCϵ inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo. Furthermore, we report that PKCϵ over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCϵ may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport. Conclusions: This study uncovers a previously unrecognized role for PKCϵ in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608–18. ©2017 AACR.

Primary myelofibrosis (PMF) is the most aggressive Philadelphia-negative myeloproliferative neoplasm. Heterogeneity in disease phenotype and outcome led to investigate the influence of germline host genetic variations on disease onset and evolution. We recently found that patients with secondary myelofibrosis are enriched in the polymorphic allele variant of the -2518 A/G SNP of the Monocyte Chemoattractant Protein-1 (MCP-1), and that this SNP correlates with disease severity (Masselli E. et al, Leukemia 2018). Here, we examined the interactive effects of MCP-1 allele variants with the baseline phenotype in PMF, and how these variants impact the disease progression. MCP-1 -2518 SNP genotyping was performed by TaqMan Predesigned SNP Genotyping Assays on DNA extracted from peripheral blood granulocytes of 201 PMF patients and 249 matched local control subjects (CTRL). One-hundred and nineteen out of 201 PMF were males (59.2%). Median age was 52y (18-80y). N.103 patients (51.2%) were pre-fibrotic PMF. Driver mutations occurred as follows: 123 (61.2%) harbored a JAK2V617F mutation, 47 (23.4%) had CALR mutations, 20 (9.9%) were triple negative and n.8 (4%) had MPL mutations. Three patients had both JAK2V617F and CALR mutation. After a median follow-up of 84.7 months, 37 patients (18.4%) died, 38 (18%) experienced blast transformation (BT) and 23 patients (11.4%) received allogeneic hematopoietic stem cell transplantation (ASCT). Median age of CTRL was 64y (28-85y), and 132/249 (53%) were males. Consistently with our previous report, PMF displayed similar genotypic and allelic frequencies of the local control population (O.R., 1.04). The G allele distribution was also similar in JAK2V617Fpos vs. JAK2V617Fneg PMF and did not correlate with the allele burden (O.R., 1.29 and 1.51, respectively). After verifying that PMF with only 1 copy of the risk allele (A/G), had a similar risk of the reference group with 0 copies (A/A), we used a recessive genetic model to compare PMF with 2 copies of the risk variant (G/G, hereby referred as the MCP-1 high-risk group) vs. A/A+A/G PMF (the MCP-1 low-risk group). Genotype-phenotype correlations with disease parameters at the time of diagnosis were performed by χ2/Fisher exact test. We found that the MCP-1 high risk group displayed a significantly higher frequency, at the time of diagnosis, of severe anemia, massive splenomegaly, elevated LDH and higher levels of hs-CRP (high-sensitivity C-reactive protein) (see Table). These results indicate that MCP-1 high-risk variant has a role in determining the disease phenotype and the pro-inflammatory microenvironment of PMF. Additionally, we assessed whether the MCP-1 high-risk variant could predispose the acquisition of risk factors in PMF. On the basis of the recessive genetic model, we found that the MCP-1 high-risk group was about four times as likely to progress to leukocytosis as was the reference group (HR, 4.16; 95% CI; 1.37 to 12.5; P=0.011). To weight the clinical relevance of our findings, we tested whether the MCP-1 high-risk variant might affect the disease progression. ASCT occurrence resulted significantly higher in the high-risk group as compared to the low-risk (HR = 3.22; 95% CI, 1.06 to 10; P=0.038). In the composite endpoint model that considered death for any cause, BT or ASCT, PMF carrying the high-risk genotype presented a shorter time to event as compared to the low-risk group (HR, 2.56; 95% CI, 1.19 to 5.55; P=0.016). In conclusion, the MCP-1 SNP appears to identify PMF patients at higher chance to present with adverse hematological features and more likely to progress rapidly to either death, BT or ASCT. Table. Disclosures No relevant conflicts of interest to declare.