Incorporation Of 32Pi Research Articles

Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine.

The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms. In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties. The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3-4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25-30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with <50% PI incorporation for the L-RWV peptides. By contrast, most H-RWV peptides displayed almost up to 100% PI incorporation. The results suggest that membrane perturbation is not the primary killing mechanism of the L-amphipathic RWV peptides, in contrast to the H-RWV peptides. Taken together, the data indicate that both types of amphipathicity may provide different ideal pharmacological properties that deserve further investigation.

An Embedded System Based on Raspberry Pi for Effective Electrocardiogram Monitoring

In recent years, there has been a growing demand for affordable and user-friendly medical diagnostic devices due to the rise in global diseases. This study focuses on the development of an embedded system based on Raspberry Pi that enables faster and more efficient monitoring of electrocardiogram (ECG). The incorporation of Raspberry Pi allows for both wireless and wired interfaces, facilitating the creation of an ECG diagnostic embedded system capable of real-time detection and immediate response to any abnormalities in heart functionality. The system presented in this research encompasses a comprehensive electronic circuit comprising analog and digital components to measure and display the ECG signal. Within the analog section, the circuit performs essential signal conditioning tasks, such as signal amplification and noise filtering, ensuring a clean signal within the desired frequency range. The entire system is powered using a power bank. The digital segment incorporates an analog-to-digital converter necessary for converting the received analog signal into a digital format compatible with Raspberry Pi. A graphical liquid-crystal display is utilized to display the measured signal. The device successfully measures ECG signals at various heart rates, capturing all crucial peaks that can be used as indicators of an individual’s health condition. By comparing the signals obtained from healthy individuals with those exhibiting heart arrhythmias, valuable insights can be gained regarding their health status. The proposed system aims to be portable, cost-effective, and user-friendly in different environments.

Abstract 608: The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML)

Abstract Background: AML is a genetically heterogenous disease with poor prognosis and new treatments are needed. MPS1 is the main kinase of the spindle assembly checkpoint (SAC), critical for segregation of chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. MPS1 inhibition has been investigated in several solid tumors but not yet in AML. Here, we demonstrate the efficacy of the small molecule MPS1 inhibitor (MPS1i) S81694 in AML cell lines, primary cells and mouse models. Materials and Methods: Cell viability, cell stress and apoptosis were assessed by MTS assays, DiOC2(3) or annexin-V modifications and PI incorporation after exposure to S81694. Cell-cycle and polyploidy were determined by cytofluorometry and FISH. Phosphorylation of MPS1 was detected in synchronized cells by immunofluorescence detecting phosphorylated Thr33/Ser37 residues. Protein modifications were studied by WB. Time-lapse microscopy was used to determine mitosis duration using cells stained with the SiR-DNA live cell probe. Bone marrow cells from patients were obtained after informed consent and incubated on a MSC feeder or in methylcellulose for colony formation assays. All animal studies were performed in accordance with the animal ethics committee's guidelines. NSG mice were sub-lethally-irradiated and injected with AML-NS8 or AML-PS patient derived xenografts. Mice were treated intravenously with S81694 or with vehicle. Results: IC50 for viability after S81694 exposure alone was &amp;lt;1000nM in a panel of 8 AML cell lines. Three cell lines had IC50 &amp;gt;1000nM: the NPM1cmut cell line OCI-AML3, OCI-AML3-TP53mut and K562. In the most sensitive cell lines, including OCI-AML2, S81694 led to inhibition of MPS1 auto-phosphorylation, induced significant cell stress and apoptosis as detected by mitochondrial membrane potential loss, phosphatidylserine exposure and PI incorporation. In these cell lines, the cell cycle was strongly affected by treatment with S81694 showing aberrant 2n/4n ploidy distribution due to SAC abrogation as well as polyploidy (8n and 16n). S81694 exposure triggered mitotic exit as shown by cyclin B1 downregulation and significantly accelerated mitosis. For treated OCI-AML2 and OCI-AML3 cells we observed induction of gamma-H2AX (ser139), p53 upregulation and downstream caspase-3 and PARP cleavage indicating that S81694 induces p53 dependent apoptosis. Furthermore, MPS1i induced downregulation of anti-apoptotic proteins MCL-1 and BCLXL. Finally, for AML patient derived blast cells cells we observed apoptosis after exposure to S81694 and significant reduction of colony number. In two AML PDX models, intravenous administration of S81694 was shown to improve significantly median survival time, as compared to control, by 28 to 41 days and by 33 to 46 days respectively. Conclusion: The MPS1i S81694 shows significant preclinical activity in vitro and in vivo in AML. Combinations with different drugs active in AML are ongoing. Citation Format: Anna Kaci, Emilie Adicéam, Etienne De Braekeleer, Marine Garrido, Jeannig Berrou, Mélanie Dupont, Hanane Djamai, Virginie Eclache, Baruchel André, Claude Gardin, Hervé Dombret, Mike Burbridge, Thorsten Braun. The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 608.

Soil phosphorus accumulation changes with decreasing temperature along a 2300 m altitude gradient

Understanding the abundance of organic P in soil is a prerequisite for predicting the effects of climate change on P dynamics and availability in cold alpine regions. We sampled plant roots (up to 40 cm depth) and soils (100 cm depth) in grasslands along a 2300 m altitude gradient (1286–3589 m above sea level, mean annual temperature (MAT) from 9 to 0.3 °C) between alpine steppes on the Loess Plateau and alpine meadows on the Tibetan Plateau. A modified Hedley P fractionation in combination with root and microbial P, alongside phosphatase activity, was used to characterize P transformations depending on climate. Both the roots and microorganisms stored more P and produced higher phosphatase activity in cold meadow than in warm steppe soils. Total inorganic P (Pi) content decreased in cold meadow (MAT < 3 °C) compared with warm steppe soils because of the decrease of moderately labile Pi (extracted with diluted HCl) in cold meadow soils. Available P (NaHCO3) and labile Pi (NaOH) contents increased upslope, whereas the recalcitrant Pi (concentrated HCl) contents remained stable at all sites down to 100 cm soil depth. Labile (NaHCO3), moderately labile (NaOH) and recalcitrant (concentrated HCl) organic P (Po) contents sharply increased in cold meadow compared with warm steppe soils. The residue P in the 0–100 cm along the temperature gradient was similar to that of all Po fractions. The Po proportion in the total P was less than 10 % at sites with a MAT above 3 °C, but sharply increased up to 50 % at sites with a MAT below 0.7 °C. The greater root and microbial P uptake versus slower Po mineralization in cold meadows than in warm steppes increase the incorporation of Pi into organic pools in cold alpine areas. This accumulation of Po forms is a mechanism to prevent P losses by leaching as inorganic forms. The increased plant available P in cold meadows was associated with the increased phosphatase activity and Po contents compared with warm steppe soils. We conclude that the on-going climate warming could stimulate P cycling more in cold meadows than in warm steppe regions because Po dominated the total soil P in cold areas.

Structural and biophysical characterizations of HIV-1 matrix trimer binding to lipid nanodiscs shed light on virus assembly

During the late phase of the HIV-1 replication cycle, the viral Gag polyproteins are targeted to the plasma membrane for assembly. The Gag-membrane interaction is mediated by binding of Gag's N-terminal myristoylated matrix (MA) domain to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The viral envelope (Env) glycoprotein is then recruited to the assembly sites and incorporated into budding particles. Evidence suggests that Env incorporation is mediated by interactions between Gag's MA domain and the cytoplasmic tail of the gp41 subunit of Env (gp41CT). MA trimerization appears to be an obligatory step for this interaction. Insufficient production of a recombinant MA trimer and unavailability of a biologically relevant membrane system have been barriers to detailed structural and biophysical characterization of the putative MA-gp41CT-membrane interactions. Here, we engineered a stable recombinant HIV-1 MA trimer construct by fusing a foldon domain (FD) of phage T4 fibritin to the MA C terminus. Results from NMR experiments confirmed that the FD attachment does not adversely alter the MA structure. Employing hydrogen-deuterium exchange MS, we identified an MA-MA interface in the MA trimer that is implicated in Gag assembly and Env incorporation. Utilizing lipid nanodiscs as a membrane mimetic, we show that the MA trimer binds to membranes 30-fold tighter than does the MA monomer and that incorporation of PI(4,5)P2 and phosphatidylserine enhances the binding of MA to nanodiscs. These findings advance our understanding of a fundamental mechanism in HIV-1 assembly and provide a template for investigating the interaction of MA with gp41CT.

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD.Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores.Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients.Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Uptake of phosphate by Synechocystis sp. PCC 6803 in dark conditions: Removal driving force and modeling

Rapid uptake of inorganic phosphate (Pi) by microalgae should occur through two processes operating in parallel: onto extracellular polymeric substances (EPS) and intracellular polymeric substances (IPS). Most previous studies focused only on overall Pi uptake and ignored the roles of EPS. We investigated the two-step removal of Pi by Synechocystis sp. PCC 6803 in dark conditions (i.e., without incorporation of Pi into newly synthesized biomass). We also developed a model to simulate both steps. Experimental results with Synechocystis confirmed that Pi in the bulk solution was removed by the two uptake mechanisms operating in parallel, but with different kinetics. All uptake rates decreased with time, and the Pi uptake rate by IPS was much higher than that by EPS at all times, but EPS had a larger maximum Pi-storage capacity -- 33–48 mgP/gCODEPS versus 15–17 mgP/gCODIPS. Synechocystis had a maximum Pi-storage capacity in the range of 22–28 mgP/g dry biomass. Protein in EPS and IPS played the key role for binding Pi, and biomass with higher protein content had greater Pi-storage capacity. Furthermore, biomass with low initial stored Pi had faster Pi-uptake kinetics, leading to more Pi removed from the bulk solution. This work lays the foundation for using microalgae as a means to remove Pi from polluted water and for understanding competition for Pi in microbial communities.

Septin-based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles.

Septins constitute a novel class of cytoskeletal proteins. Budding yeast septins self-assemble into non-polar filaments bound to the inner plasma membrane through specific interactions with l-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Biomimetic in vitro assays using giant unilamellar vesicles (GUVs) are relevant tools to dissect and reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. This report is dedicated to optimize the incorporation of PI(4,5)P2 lipids into GUVs by probing the proteins-PI(4,5)P2 GUVs interactions. We show that the interaction between budding yeast septins and PI(4,5)P2 is more specific than using usual reporters (phospholipase Cδ1). Septins have thus been chosen as reporters to probe the proper incorporation of PI(4,5)P2 into giant vesicles. We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction resulting from an optimal PI(4,5)P2 incorporation for which, we have optimized the growth conditions. Finally, we have shown that PI(4,5)P2 GUVs have to be used within a few hours after their preparation. Indeed, over time, PI(4,5)P2 is expelled from the GUV membrane and the PI(4,5)P2 concentration in the bilayer decreases.

Understanding docetaxel and cabazitaxel modes of action in prostate cancer.

e23149 Background: Despite the approval of several new agents, taxanes remains the main treatment with survival benefits for castration-refractory metastatic prostate cancer/mCRPC. Still their mechanisms of action and therapeutic efficacy remain incompletely characterized. In the present study, we proposed to compare docetaxel/Doc and cabazitaxel/Cab, delivered as monotherapy or in combination with the anti-angiogenic and anti-tumoral Pigment Epithelium-Derived Factor/PEDF, on CRPC cells in vitro and in vivo. Methods: CRPCcells were assessed for cell growth, cell cycle, and apoptosis under taxane/control treatment by cytotoxicity, PI incorporation and TUNEL assays. CL1 cells that express PEDF/control were used in vivo. Tumor cells were injected s.c. into CB17-SCID mice. After two weeks, mice were randomized into groups: 1) Placebo; 2) Doc 5mg/kg i.p. d4; and 3) Cab 5-1-0.5-0.1mg/kg i.p. d4, d1-7, d2, daily. To assess the anti-tumor effect of the combination, tumor cell migration and phagocytosis were measured by Boyden chamber and confocal microscopy analyses of CL1-RAW264.7 macrophages co-cultures. Results: Cab was more cytotoxic than Doc in all the cell lines tested.This effect was concomitant to increased cell death, but was not due to autophagy or necrosis. Inversely, we showed that apoptosis was superior in Cab-treated cells than in Doc treatment. In vivo, while 0.5 and 0.1 mg/kg Cab did not improve PEDF efficacy on tumor growth, 1mg/kg was very toxic. In contrast, PEDF combined with 5mg/kg Cab lead to stabilization of the disease and was also found to be drastically more efficient than PEDF/Doc in delaying the disease. In vitro, PEDF/Cab inhibited tumor cell migration at a significant superior level compared to PEDF/Doc. Finally, tumor cells phagocytosis was induced in PEDF/Cab suggesting that the combination may target macrophages within the tumor microenvironment. Conclusions: Our data demonstrated the greater anti-tumor efficacy of Cab compared to Doc. They also insist on the fact that PEDF/Cab could be used as a novel combined therapy for CRPC, and emphasize on the importance in evaluating the cytotoxicity of the novel combination tested and investigating the role of the tumor microenvironment in the anti-tumor effect.

Intense biological phosphate uptake onto particles in subeuphotic continental margin waters

AbstractElucidating the processes that affect particulate phosphorus (P) export from the euphotic zone and burial in sediments is important for models of global phosphorus, nitrogen, and carbon cycling. We investigated dissolved inorganic Pi incorporation into particles (&gt;0.2 µm) in the subeuphotic zone and benthic boundary layer of high‐productivity Mauritanian and Namibian shelf waters, using 33PO43− tracer experiments combined with a sequential chemical extraction analysis. Pi uptake (5.4 to 19.9 nmol P L−1 d−1) by particulate matter was biologically mediated (~50% into the organic fraction) and similar to estimated rates of heterotrophic growth. Thus, a substantial fraction of Pi must be recycled through a particle‐associated microbial pool. Rapid adsorption of 33P in the anoxic waters of Namibia indicated the additional existence of a large pool of surface exchangeable P. Particle‐associated Pi recycling and adsorption may influence the export flux and ultimate fate of particle bound P in continental shelf waters.

A new high-performance ionic polymer–metal composite based on Nafion/polyimide blends

For the first time, we report ion-exchange membranes based on Nafion and polyimide (PI, Kapton) blends to fabricate ionic polymer–metal composites (IPMCs). Polyamic acid [PAA, poly(pyromellitic dianhydride-co-4,4′-oxydianiline), as a precursor of PI] solution was blended with Nafion solution using physical blending method to provide PAA–Nafion blend membrane. This work demonstrates that, by simple physical blending method, the thermal and mechanical properties of Nafion can be improved while maintaining the excellent actuating performance. After thermal imidization, PAA converted into PI, resulting in PI–Nafion blend membrane. Optimum conditions to cast PAA–Nafion blends and thermal imidization have been established, and blend membranes with PI wt% of 6, 12, 18, and 30 were prepared. Fourier transform infrared spectroscopy confirmed the incorporation of PI in the Nafion matrix. Thermal decomposition unique to the PI became more noticeable as the content of PI increased, which was measured by thermogravimetric analysis. Dynamic mechanical analysis showed that the storage modulus (E′) increased as a function of PI content while loss modulus (E″) exhibited only a minor change, which resulted in the decrease in the damping properties (tan δ). The blend membranes were fabricated into IPMCs by deposition of platinum electrode onto the membrane surface through electroless plating process. Among tested, NPI-18 IPMC actuator, which has 18 wt% of PI in Nafion, showed comparable electromechanical performance to the commercially available Nafion 117 IPMC actuator.

The Interplay between Calmodulin and Membrane Interactions with the Pleckstrin Homology Domain of Akt

The Akt protein, a serine/threonine kinase, plays important roles in cell survival, apoptosis, and oncogenes. Akt is translocated to the plasma membrane for activation. Akt-membrane binding is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). It has been shown that Akt activation in breast cancer cells is modulated by calmodulin (CaM). However, the molecular mechanism of the interplay between CaM and membrane binding is not established. Here, we employed nuclear magnetic resonance (NMR) and biochemical and biophysical techniques to characterize how PI(3,4,5)P3, CaM, and membrane mimetics (nanodisc) bind to Akt(PHD). We show that PI(3,4,5)P3 binding to Akt(PHD) displaces the C-terminal lobe of CaM but not the weakly binding N-terminal lobe. However, binding of a PI(3,4,5)P3-embedded membrane nanodisc to Akt(PHD) with a 103-fold tighter affinity than PI(3,4,5)P3 is able to completely displace CaM. We also show that Akt(PHD) binds to both layers of the nanodisc, indicating proper incorporation of PI(3,4,5)P3 on the nanodisc surface. No detectable binding has been observed between Akt(PHD) and PI(3,4,5)P3-free nanodiscs, demonstrating that PI(3,4,5)P3 is required for membrane binding, CaM displacement, and Akt activation. Using pancreatic cancer cells, we demonstrate that inhibition of Akt-CaM binding attenuated Akt activation. Our findings support a model by which CaM binds to Akt to facilitate its translocation to the membrane. Elucidation of the molecular details of the interplay between membrane and CaM binding to Akt may help in the development of potential targets to control the pathophysiological processes of cell survival.

Phosphorus dynamics in biogeochemically distinct regions of the southeast subtropical Pacific Ocean

The southeast subtropical Pacific Ocean was sampled along a zonal transect between the coasts of Chile and Easter Island. This remote area of the world’s ocean presents strong gradients in physical (e.g., temperature, density and light), chemical (e.g., salinity and nutrient concentrations) and microbiological (e.g., cell abundances, biomass and specific growth rates) properties. The goal of this study was to describe the phosphorus (P) dynamics in three main ecosystems along this transect: the upwelling regime off the northern Chilean coast, the oligotrophic area associated with the southeast subtropical Pacific gyre and the transitional area in between these two biomes. We found that inorganic phosphate (Pi) concentrations were high and turnover times were long (>210nmoll−1 and >31d, respectively) in the upper water column, along the entire transect. Pi uptake rates in the gyre were low (euphotic layer integrated rates were 0.26mmolm−2d−1 in the gyre and 1.28mmolm−2d−1 in the upwelling region), yet not only driven by decreases in particle mass or cell abundance (particulate P- and cell- normalized Pi uptake rates in the euphotic layer were ∼1–4 times and ∼3–15 times lower in the gyre than in the upwelling, respectively). However these Pi uptake rates were at or near the maximum Pi uptake velocity (i.e., uptake rates in Pi amended samples were not significantly different from those at ambient concentration: 1.5 and 23.7nmoll−1d−1 at 50% PAR in the gyre and upwelling, respectively). Despite the apparent Pi replete conditions, selected dissolved organic P (DOP) compounds were readily hydrolyzed. Nucleotides were the most bioavailable of the DOP substrates tested. Microbes actively assimilated adenosine-5′-triphosphate (ATP) leading to Pi and adenosine incorporation as well as Pi release to the environment. The southeast subtropical Pacific Ocean is a Pi-sufficient environment, yet DOP hydrolytic processes are maintained and contribute to P-cycling across the wide range of environmental conditions present in this ecosystem.

A Novel Antisense Long Noncoding RNA Modulates NR4A1 Protein Level in Myeloid Malignancies

Introduction: The advent of whole transcriptome sequencing technologies revealed that most of the human genome is transcribed, producing a large repertoire of long noncoding RNAs (lncRNAs). They are known to participate in epigenetic regulation at different levels and alter mRNA stability of coding genes, emerging as key players in carcinogenesis. In the present study, we identified a novel unspliced long noncoding RNA that is transcribed from the opposite strand on the NR4A1 gene locus. The tumor suppressor NR4A1 has been associated with the regulation of apoptosis and proliferation in various tumors and shows a reduced expression in myelodysplastic syndrome (MDS). Additionally, the abrogation of this locus in murine models caused the development of acute myeloid leukemia (AML). We hypothesized that lncNR4A1 plays an important role in hematopoietic malignancy pathogenesis and possibly regulates its coding counterpart.Methods: Expression of lncNR4A1 and its sense gene NR4A1 were evaluated in CD34+ cells from bone marrow of normal donors (n=7) and patients (n= 27; MDS= 14 [low‐risk=8, high‐risk=6], AML with myelodysplastic related changes [AML‐MRC]=4 and de novo AML=9) by qRT-PCR. Results were expressed as mean (minimum-maximum) after appropriate statistical analysis. The orientation and full-length of lncNR4A1 were confirmed by strandspecific RT-PCR, RACE-PCR and sequencing techniques. For functional analysis, lncNR4A1 was inhibited with a lentiviral vector system in U937 cells. Apoptosis was evaluated by Annexin‐V/PI and the cell cycle analysis was performed applying PI incorporation by flow cytometer analysis. Mitochondrial activity was evaluated with MTT assays. Western blot was used to measure the NR4A1 protein expression and for monitoring key proteins in the cell cycle signaling pathway.Results:NR4A1 mRNA was significantly decreased in bone marrow/CD34+ from patients with MDS and AML compared to healthy donors (p<0.05), consistent with previous studies. In contrast, the long noncoding expression was significantly increased in patients with low-risk MDS (4.60[0.20-10.05]) and high-risk MDS (8.40[2.54-16.87]; p=0.008) compared to healthy donors (1.22[0.02-4.11]). Next, we examined the pattern of expression of the non-coding transcript compared with NR4A1 gene expression from each MDS patients and healthy donors. Interestingly the results showed higher levels of noncoding expression when compared to NR4A1 gene expression in MDS patients, however, noncoding expression was less abundant compared with coding gene expression in all of healthy donors samples (low-risk MDS 3.55[-6.25 to 14.54]; p=0.020 and high-risk MDS 17.43[2.15 to 50.17] p=0.002; donors -13.06 [-33.3 to -3.22]). The strand-oriented RT-PCR show that lncNR4A1 is transcribed in the antisense direction relative to protein-coding mRNA encoded in NR4A1 locus in two different leukemia cell lines. Using a combined approach involving RACE with primer-walking PCR and sequencing, we extend the sequence resulting in a full-length lncNR4A1 transcript of 5581 nt, overlapping the exons 3,4,5 and 6 of the coding gene. The knockdown of lncNR4A1 with shRNA did not affect the abundance of NR4A1 mRNA, but unexpectedly increased the NR4A1 protein expression levels. Noncoding depletion reduced significantly the cell viability compared with control cells (p=0.007). In addition to reducing cell proliferation, lncNR4A1 sh-mediated depletion induced the increase on the sub-G1 percentage population cells with a subsequent decrease of DNA synthesis S phase (p=0.027) compared with control cells. Moreover, lncNR4A1 knockdown increased p21 protein levels which correlates with G1 phase cell arrest, and with blocking the S phase entrance and cell growth. We also detected a slight Cyclin A down-regulation after noncoding inhibition which explains the reduction of G1-S transition. No significant difference was found in cell death between sh-noncoding and control cells.Conclusions: Our results showed a post-transcriptional modulation of the NR4A1 by a novel long noncoding RNA and suggested a putative oncogenic role of lncNR4A1 in hematologic malignancy. Since NR4A1 is a potent tumor suppressor for leukemia and MDS, the inhibition of lncNR4A1 could emerge as a new therapeutic strategy by upregulation of NR4A1 protein. DisclosuresNo relevant conflicts of interest to declare.

ID: 228: Irisin – A newly discovered adipomiokine – Impairs growth and progression of breast cancer MDA-MB-231 cell line

Purpose: The beneficial effect of physical activity in both prevention and therapy of breast cancer has been proven by numerous studies; however, the mechanism of this phenomenon is still unclear. Considering the above and recent studies reporting irisin as a new factor released during physical activity, we determined its effect on tumor growth and progression. Method: The study was carried out on malignant human breast carcinoma cell line (MDA-MB-231) cultured in a medium supplemented with irisin (0–100 nM). Relative gene expression was performed using Real-time PCR. Apoptosis was measured using Annexin V kit, cell growth was quantified by PI incorporation or soft agar colony formation assay. Moreover, CytoSelect migration and invasion assay was performed. Results: The inhibition of both anchorage-dependent and anchorage-independent cell growth accompanied by a increase in cell apoptosis in a dose- and time-dependent manner was observed. The cell migration and invasion through the extracellular matrix were impaired by irisin at 50 or 100 nM. Increased expression of genes associated with the inhibition of tumor growth and its progression was found (CHEK2, ATM, BRCA2, E2F4, HDAC2 and 9 or MRF11A) when cells were treated with high irisin concentration (100 nM). Also, reduction in the expression of protooncogene PLK1, as well as E2F1, GADD45B, HDAC5 and 6, RAD51 or SMARCA2 was noted. Conclusion: We show here that irisin – as a mediator of exercise – can be a potential anti-tumor agent. Further studies are still needed to establish the role of irisin in the development and progression of breast cancer.

Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3β pathway

Cerebral ischemia is the third most common cause of death and a major cause of disability worldwide. Beyond a shortage of essential metabolites, ischemia triggers many interconnected pathophysiological events, including excitotoxicity, oxidative stress, inflammation and apoptosis. Here, we investigated the neuroprotective mechanisms of JM-20, a novel synthetic molecule, focusing on the phosphoinositide-3-kinase (PI3K)/Akt survival pathway and glial cell response as potential targets of JM-20. For this purpose, we used organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD) to achieve ischemic/reperfusion damage in vitro. Treatment with JM-20 at 0.1 and 10 μM reduced PI incorporation (indicative of cell death) after OGD. OGD decreased the phosphorylation of Akt (pro-survival) and GSK 3β (pro-apoptotic), resulting in respective inhibition and activation of these proteins. Treatment with JM20 prevented the reduced phosphorylation of these proteins after OGD, representing a shift from pro-apoptotic to pro-survival signaling. The OGD-induced activation of caspase-3 was also attenuated by JM-20 treatment at 10 μM. Moreover, in cultures treated with JM-20 and exposed to OGD conditioning, we observed a decrease in activated microglia, as well as a decrease in interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α release into the culture medium, while the level of the anti-inflammatory IL-10 increased. GFAP immunostaining and IB4 labeling showed that JM-20 treatment significantly augmented GFAP immunoreactivity after OGD, when compared with cultures exposed to OGD only, suggesting the activation of astroglial cells. Our results confirm that JM-20 has a strong neuroprotective effect against ischemic injury and suggest that the mechanisms involved in this effect may include the modulation of reactive astrogliosis, as well as neuroinflammation and the anti-apoptotic cell signaling pathway.

Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration

Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL+-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL+-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL+-cells/mm2 in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL+ cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V+-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

BRD4 Short Isoform Is a Myelodysplasia Prognostic Marker Involved in Inappropriate DNA Damage Response

INTRODUCTION: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. BRD4 is a BET member of bromodomain-containing proteins, known as epigenome readers, exerting key roles in chromatin remodeling and transcriptional regulation. BRD4 gene encodes two major isoforms, short (BRD4S) and long (BRD4L). Recently, Floyd SR et al (Nature, 2013) described BRD4S as an endogenous inhibitor of the DNA damage response (DDR). Under normal conditions, damaged DNA induces histone phosphorylation of H2AX at Ser 139 (γ-H2AX) and activates a protective signaling network that blocks cell cycle and recruits DNA repair factors. Despite having been described as a therapeutic target in acute myeloid leukemia (AML) by Zuber J et al (Nature, 2011), BRD4 has never been studied in myelodysplastic syndrome (MDS) and its role in the pathogenesis is currently unknown.AIMS: To evaluate BRD4L and BRD4S expression in MDS and AML patients, correlating with clinical data, progression and survival. We also explored the BRD4 role in DDR signaling using human leukemia cell line models.MATERIAL AND METHODS: Diagnostic total bone marrow (BM) samples from 24 healthy donors (HD) and 99 patients, including 48 MDS (22 higher-risk MDS and 36 lower-risk MDS) and 51 AML (16 of them with MDS-related changes, AML-MRC), were collected. We also isolated CD34+ cells from 7 HD, 5 de novo AML, 4 AML-MRC and 14 MDS (6 higher-risk and 8 lower-risk MDS). BRD4L and BRD4S gene expressions were assessed through q-PCR and expressed as median (minimum-maximum). MDS patients were stratified according IPSS, WHO classification, R-IPSS and cytogenetic risk. Samples were age-adjusted when significant differences were observed, using ANOVA and Tukey’s test. Progression-free and overall survival curves were estimated by Kaplan-Meier method and were analyzed by the Wilcoxon´s test and Cox regression. JQ1, a specific BRD4 inhibitor, was kindly provided by James Bradner. A panel of human myeloid leukemia cell lines (KG1a, HEL, HL60, U937) in exponential growth was treated with increasing doses of JQ1 for 48 hours and cell growth (MTT colorimetric assay), apoptosis (annexin-V/PI) and cell cycle (flow cytofluorometric analysis detecting nuclear PI incorporation) were evaluated. We also determined the expression of p-γH2AX (DDR signaling) by western blot, after 12 hours of JQ1 treatment.RESULTS: A higher expression of BRD4S was observed in total BM cells from AML (4.01 [0.33-2.58], P=.01) and MDS patients (4.21 [0.01-56.17], P=.01) compared with HD (2.11 [0.04-10.32], P=.01). When stratified according WHO classification, AML-MRC (4.5 [0.33-25.22], P=.04) and higher-risk MDS (4.66 [0.17-56.17], P=.04) subgroups showed higher BRD4S expression. In CD34+ cells, BRD4S expression was increased in de novo AML (0.28 [0.21-0.45]) compared with lower-risk MDS (0.02 [0.00-0.44], P=.01). BRD4L mRNA expression was not modulated in total BM and CD34+ cells from any subgroup. With median follow-up time of 34.4 months, we found that higher BRD4S gene expression was a worse prognostic factor for MDS transformation and survival, along with IPSS, R-IPSS, low hemoglobin (less than 10g/dL) and higher BM blast percentage. After multivariate analysis, BRD4S gene expression and higher-risk (very high, high and intermediate) R-IPSS remained as independent prognostic factors for MDS progression and overall survival. KG1a and U937 cells showed greater resistance to JQ1, with lower apoptosis rate and proliferation (IC50 not reached, over 3000nM), whereas HEL and HL60 were more responsive (IC50 under 800nM in both cell lines). JQ1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and apoptosis and caused a progressive increase in histone phosphorylation of γ-H2AX, indicating activation of DDR signaling.CONCLUSIONS: MDS is a clonal myeloid neoplasm characterized by profound epigenetic modifications. Our data establishes BRD4S as a novel MDS prognostic factor, related to aggressive phenotype, higher progression rate and shorter survival. Biologically, BRD4S plays a role in the inappropriate DNA Damage Response of MDS, favoring the disease towards genetic instability and clonal evolution. DisclosuresNo relevant conflicts of interest to declare.

New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.