High Inhibitory Concentration Research Articles

The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition

The G2019S mutation in LRRK2 is one of the most common known genetic causes of neurodegeneration and Parkinson disease (PD). LRRK2 mutations are thought to enhance LRRK2 kinase activity. Efficacious small molecule LRRK2 kinase inhibitors with favorable drug properties have recently been developed for pre-clinical studies in rodent models, and inhibitors have advanced to safety trials in humans. Rats that express human G2019S-LRRK2 protein and G2019S-LRRK2 knock-in mice provide newly characterized models to better understand the ostensible target for inhibitors. Herein, we explore the relationships between LRRK2 kinase inhibition in the brain and the periphery to establish the link between LRRK2 kinase activity and protein stability, induction of lysosomal defects in kidney and lung, and how G2019S-LRRK2 expression impacts these phenotypes. Using a novel ultra-sensitive scalable assay based on protein capillary electrophoresis with LRRK2 kinase inhibitors included in-diet, G2019S-LRRK2 protein was resilient to inhibition compared to wild-type (WT)-LRRK2 protein, particularly in the brain. Whereas WT-LRRK2 kinase activity could be completed blocked without lowering LRRK2 protein levels, higher inhibitor concentrations were necessary to fully reduce G2019S-LRRK2 activity. G2019S-LRRK2 expression afforded robust protection from inhibitor-induced kidney lysosomal defects, suggesting a gain-of-function for the mutation in this phenotype. In rodents treated with inhibitors, parallel measurements of phospho-Rab10 revealed a poor correlation to phospho-LRRK2, likely due to cells that express Rab10 but poorly express LRRK2 in heterogenous tissues and cell isolates. In summary, our results highlight several challenges associated with the inhibition of the G2019S-LRRK2 kinase that might be considered in initial clinical efforts.

Detection of a novel, primate-specific 'kill switch' tumor suppression mechanism that may fundamentally control cancer risk in humans: an unexpected twist in the basic biology of TP53.

The activation of TP53 is well known to exert tumor suppressive effects. We have detected a primate-specific adrenal androgen-mediated tumor suppression system in which circulating DHEAS is converted to DHEA specifically in cells in which TP53 has been inactivated. DHEA is an uncompetitive inhibitor of glucose-6-phosphate dehydrogenase (G6PD), an enzyme indispensable for maintaining reactive oxygen species within limits survivable by the cell. Uncompetitive inhibition is otherwise unknown in natural systems because it becomes irreversible in the presence of high concentrations of substrate and inhibitor. In addition to primate-specific circulating DHEAS, a unique, primate-specific sequence motif that disables an activating regulatory site in the glucose-6-phosphatase (G6PC) promoter was also required to enable function of this previously unrecognized tumor suppression system. In human somatic cells, loss of TP53 thus triggers activation of DHEAS transport proteins and steroid sulfatase, which converts circulating DHEAS into intracellular DHEA, and hexokinase which increases glucose-6-phosphate substrate concentration. The triggering of these enzymes in the TP53-affected cell combines with the primate-specific G6PC promoter sequence motif that enables G6P substrate accumulation, driving uncompetitive inhibition of G6PD to irreversibility and ROS-mediated cell death. By this catastrophic ‘kill switch’ mechanism, TP53 mutations are effectively prevented from initiating tumorigenesis in the somatic cells of humans, the primate with the highest peak levels of circulating DHEAS. TP53 mutations in human tumors therefore represent fossils of kill switch failure resulting from an age-related decline in circulating DHEAS, a potentially reversible artifact of hominid evolution.

P-351 - Characterization of new stress response genes in Listeria monocytogenes

In this work we characterize lisA and lisT, two new genes involved on the stress response of Listeria. These genes are specific of the pathogenic L. monocytogenes but not in the close related non-pathogenic L. innocua, suggesting they may play a role in virulence of this intracellular bacterium. lisA and lisT encode two hypothetic small proteins of unknow function that show characteristics of type II toxin/antitoxin (TA) systems. Such TA systems are increasingly recognized as novel important regulators of virulence. Here we show that overexpression of LisT, but not of LisA, revealed to be toxic in E. coli cells. Furthermore, LisT presents an alternative start codon suggesting a tight translational control of its expression. LisA and List are new genes involved in the bacterial response to antibiotics. In the presence of high concentrations of translation inhibitors, namely chloramphenicol, kanamycin or erythromycin, the mRNA levels of these genes are strongly upregulated. The role of these genes in the bacterial persistence phenomenon, a serious clinical threat to successful treatment of severe infections, was further investigated. Overall, LisA/LisT is suggested to be a novel type II toxin/antitoxin system with relevance for the virulence of Listeria monocytogenes.

Inhibiting and Remodeling Toxic Amyloid-Beta Oligomer Formation Using a Computationally Designed Drug Molecule That Targets Alzheimer's Disease.

Alzheimer's disease (AD) is rapidly reaching epidemic status among a burgeoning aging population. Much evidence suggests the toxicity of this amyloid disease is most influenced by the formation of soluble oligomeric forms of amyloid β-protein, particularly the 42-residue alloform (Aβ42). Developing potential therapeutics in a directed, streamlined approach to treating this disease is necessary. Here we utilize the joint pharmacophore space (JPS) model to design a new molecule [AC0107] incorporating structural characteristics of known Aβ inhibitors, blood-brain barrier permeability, and limited toxicity. To test the molecule's efficacy experimentally, we employed ion mobility mass spectrometry (IM-MS) to discover [AC0107] inhibits the formation of the toxic Aβ42 dodecamer at both high (1:10) and equimolar concentrations of inhibitor. Atomic force microscopy (AFM) experiments reveal that [AC0107] prevents further aggregation of Aβ42, destabilizes preformed fibrils, and reverses Aβ42 aggregation. This trend continues for long-term interaction times of 2days until only small aggregates remain with virtually no fibrils or higher order oligomers surviving. Pairing JPS with IM-MS and AFM presents a powerful and effective first step for AD drug development. Graphical Abstract.

Inhibition of α‐Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends

AbstractMisfolding of the protein α‐synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh‐β) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh‐β concentrations (IC50=850 nm) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.

Inhibition of α-Synuclein Amyloid Fibril Elongation by Blocking Fibril Ends.

Misfolding of the protein α-synuclein (αSyn) into amyloid fibrils plays a central role in the development of Parkinson's disease. Most approaches for the inhibition of αSyn fibril formation are based on stabilizing the native monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. In this work, we designed an inhibitor (Inh-β) that selectively binds the growing ends of αSyn fibrils and creates steric hindrance for the binding of monomeric αSyn. This approach permits the inhibition of fibril formation at Inh-β concentrations (IC50 =850 nm) much lower than the concentration of monomeric αSyn. We studied its kinetic mechanism in vitro and identified the reactions that limit inhibition efficiency. It is shown that blocking of αSyn fibril ends is an effective approach to inhibiting fibril growth and provides insights for the development of effective inhibitors of αSyn aggregation.

Inhibitors degradation and microbial response during continuous anaerobic conversion of hydrothermal liquefaction wastewater

One critical challenge of hydrothermal liquefaction (HTL) is its complex aqueous product, which has a high concentration of organic pollutants (up to 100gCOD/L) and diverse fermentation inhibitors, such as furfural, phenolics and N-heterocyclic compounds. Here we report continuous anaerobic digestion of HTL wastewater via an up-flow anaerobic sludge bed reactor (UASB) and packed bed reactor (PBR). Specifically, we investigated the transformation of fermentation inhibitors and microbial response. GC–MS identified the complete degradation of furfural and 5-hydroxymethylfurfural (5-HMF), and partial degradation (54.0–74.6%) of organic nitrogen and phenolic compounds, including 3-hydroxypyridine, phenol and 4-ethyl-phenol. Illumina MiSeq sequencing revealed that the bacteria families related to detoxification increased in response to the HTL aqueous phase. In addition, the increase of acetate-oxidizing bacteria in UASB and acetogens in PBR showed a strengthened acetogenesis. As for the archaeal communities, an increase in hydrogenotrophic methanogens was observed. Based on GC–MS/HPLC and microbial analysis, we speculate that dominant fermentation inhibitors were transformed into intermediates (Acetyl-CoA and acetate), further contributing to biomethane formation.

Comparison of Meconium DNA Extraction Methods for Use in Microbiome Studies.

The establishment of human gut microbiota commences initially in utero. Meconium—the first fecal material passed after birth—can be used to study fetal gut contents; however, processing meconium samples for microbiome studies presents significant technical challenges. Meconium hosts a low biomass microbiome, is tar-like in texture and contains high concentrations of PCR inhibitors. This study aimed to evaluate four different DNA extraction methods to elucidate the most effective method for bacterial DNA recovery and sequencing analysis from first-pass meconium. Samples from five infants were collected and processed using the following extraction kits: (1) Qiagen QIAamp DNA Stool Mini (QS); (2) Qiagen QIAamp DNA Microbiome (QM); (3) MoBio PowerSoil (PS); (4) MoBio MagAttract PowerMicrobiome (PM). Additionally, Kit PM was employed with a double inhibitor removal treatment (IRT) step (PM2). Bacterial DNA recovery was assessed by qPCR. Any PCR inhibition in samples was measured by spiking DNA eluates with 0.1 ng of pure Streptococcus agalactiae (GBS) DNA followed by qPCR quantitation. Kit PM yielded the highest average total DNA yield (79.3 ng per gram of meconium). Samples extracted with kit PS had the highest detectable levels of 16S rRNA gene by qPCR. The ability of each kit to overcome PCR inhibition varied, with qPCR on GBS-spiked DNA from kits QS, QM, PS, and PM recovering 87.1, 91.0, 88.8, and 37.9% GBS DNA, respectively. Double IRT improved the performance of kit PM, increasing GBS recovery to 56.5%. However, once DNA yield was normalized to the level recovered with the other kits 100% of GBS DNA was detected, suggesting that levels of PCR inhibitors are related to DNA yield from kit PM. Ion Torrent 16S rRNA gene sequencing revealed a high level of inter-kit variation in meconium microbiome structure. In particular, kit QM showed a bias toward extracting Firmicute DNA, while the other kits extracted primarily Proteobacterial DNA. Choice of extraction kit greatly impacts on the ability to extract and detect bacterial DNA in meconium and on the microbiome community structure generated from these samples.

Mechanisms of Modulation of Calcium Phosphate Pathological Mineralization by Mobile and Immobile Small-Molecule Inhibitors.

Potential pathways for inhibiting crystal growth are via either disrupting local microenvironments surrounding crystal-solution interfaces or physically blocking solute molecule attachment. However, the actual mode of inhibition may be more complicated due to the characteristic time scale for the inhibitor adsorption and relaxation to a well-bound state at crystal surfaces. Here we demonstrate the role of citrate (CA) and hydroxycitrate (HCA) in brushite (DCPD, CaHPO4·2H2O) crystallization over a broad range of both inhibitor concentrations and supersaturations by in situ atomic force microscopy (AFM). We observed that both inhibitors exhibit two distinct actions: control of surface crystallization by the decrease of step density at high supersaturations and the decrease of the [1̅00]Cc step velocity at high inhibitor concentration and low supersaturation. The switching of the two distinct modes depends on the terrace lifetime, and the slow kinetics along the [1̅00]Cc step direction provides specific sites for the newly formed dislocations. Molecular modeling shows the strong HCA-crystal interaction by molecular recognition, explaining the AFM observations for the formation of new steps and surface dissolution along the [101]Cc direction due to the introduction of strong localized strain in the crystal lattice. These direct observations highlight the importance of the inhibitor coverage on mineral surfaces, as well as the solution supersaturation in predicting the inhibition efficacy, and reveal an improved understanding of inhibition of calcium phosphate biomineralization, with clinical implications for the full therapeutic potential of small-molecule inhibitors for kidney stone disease.

Ginger extract as green corrosion inhibitor of mild steel in hydrochloric acid solution

Ginger extract as corrosion inhibitor from natural resources was studied to prevent corrosion of mild steel in acid media. Ginger rhizome was extracted to produce green corrosion inhibitor (G-1) while ginger powder bought at supermarket was also extract to form green corrosion inhibitor (G-2). Effectiveness of inhibitor in preventing corrosion process of mild steel was studied in 1.0 M of hydrochloric acid. The experiment of weight loss method and polarization technique were conducted to measure corrosion rate and inhibition efficiency of mild steel in solution containing 1.0 M of hydrochloric acid with various concentration of inhibitor at room temperature. The results showed that, the rate of corrosion dropped from 8.09 mmpy in solution containing no inhibitor to 0.72 mmpy in solution containing 150g/l inhibitor while inhibition efficiency up to 91% was obtained. The polarization curve in polarization experiments shows that the inhibition efficiency is 86% with high concentration of inhibitor. The adsorption of ginger extract on the surface of mild steel was observed by using optical microscope and the characterization analysis was done by using pH measurement method. When high concentration of green inhibitor in the acid solution is used, the pH at the surface of steel is increasing.

Quantitative Evaluation of Dissociation Mechanisms in Methylorange and Methylred

The inhibition of mild steel corrosion in acidic medium inhibited by amlodipine at room temperature (30 o C) was monitored via the linear polarization resistance electrochemical technique using a variable voltage, DC power supply (AC mV metre, 2channel model GVT 427). The concentration of the acid medium was maintained at 0.1M HCl throughout the experiment. The inhibition efficiency was found to increase with increase in the concentration of the drug in the corrosive medium. The highest polarization resistance (Rp) (1083Ωcm 2 ) was obtained at the highest inhibitor concentration (10.0×10 -3 M) while the lowest Rp (567Ωcm 2 ) was recorded against the lowest inhibitor concentration (2.5×10 -3 M). The adsorption mode of the drug was found to be by monolayer chemisorptions having found the experimental data obeyed the Langmuir adsorption isotherm.

High concentrations of dipeptidyl peptidase-4 inhibitors suppressed lipopolysaccharide-induced inflammatory cytokine expression in U937 cells

High concentrations of dipeptidyl peptidase-4 inhibitors suppressed lipopolysaccharide-induced inflammatory cytokine expression in U937 cells

Abstract A117: Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells

Abstract ATM is a master regulator of the DNA damage response (DDR) elicited by double-strand breaks (DSB). After DNA DSB, ATM controls cell fate via cell cycle arrest, apoptosis, and DNA damage repair. Numerous tumor types have ATM gene loss-of-function mutations, resulting in genomic instability. Most knowledge on the effect of ATM loss on DDR and tumor therapeutic response is from ataxia-telangiectasia (A-T) patients and ATM knockout models. A distinct and more deleterious phenotype has been proposed for kinase-dead (KD) ATM mutants that retain ATM protein expression. Inhibition of ATM activity does not fully mimic ATM loss; therefore, KD ATM mutants may be better predictors of response to ATM inhibitors and chronic effects of ATM inhibition. Using an inducible lentiviral CRISPR/Cas9 genome-editing system, we generated in-frame ATM mutated clonal lines derived from a bladder cancer PDX model (BLX1), via targeting of ATM exon 57, which maps to the second exon in the kinase domain. CRISPR-edited clones consistently exhibited reduced phosphorylation of Kap1 (S824) but not CHK2, p53 or NBS1 when challenged with DSB-inducing agents such as ionizing radiation (IR) and etoposide. Similar results were obtained in unedited cells treated with the ATM inhibitor KU55933, confirming that changes observed are ATM-dependent and suggesting that reduced Kap1 phosphorylation is a robust readout of compromised ATM activity in response to DSB-inducing therapies. Radiosensitivity is a hallmark of the A-T syndrome and the CRISPR-edited clones had increased sensitivity to DSB-inducing agents including IR, bleomycin, etoposide, and doxorubicin but not to S-phase drugs gemcitabine and camptothecin in a 10-day colony formation assay. ATM deficiency increased the dependency of cancer cells on ATM-independent repair mechanisms. Indeed, short-term (4-day) proliferation assays demonstrated the synthetic lethality of KD ATM with ATR inhibitors (AZD6738, VE821), Chk1 inhibitors (MK8776, LY2606368), as well as high concentrations of PARP inhibitors (Olaparib, BMN673) but not the DNAPK inhibitor (NU7441). KD ATM clones did not have increased response to cisplatin as a single agent except at high concentrations (>10 µM); the combination of low-concentration cisplatin (<1 µM) and an ATR inhibitor had a greater effect in KD ATM clones compared to unedited cells, demonstrating increased dependence on ATR. In summary, CRISPR-Cas9 editing of the ATM kinase domain was a useful tool for characterizing ATM loss-of-function effects, identifying targets to resensitize ATM mutants and elucidating compensatory mechanisms driven by chronic ATM loss. Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Lara H. El Touny, John Connelly, Curtis Hose, Anne Monks, Dianne Newton, Luke Stockwin, Melinda Hollingshead, Ralph Parchment, James H. Doroshow, Beverly A. Teicher, Annamaria Rapisarda. Modeling ATM loss of function via CRISPR-Cas9 as a predictive tool for therapeutic responses in cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A117.

Effect of an angiotensin-converting-enzyme inhibitor on the plasma concentration of cytokines and vasoactive molecules in patients with coronary heart disease and hypertension

To investigate the plasma concentrations of cytokines and vasoactive molecules in patients with coronary heart disease (CHD) in the presence of hypertension in relation to the angiotensin-converting-enzyme (ACE) inhibitor level reflecting the degree of renin-angiotensin-aldosterone system (RAAS) inhibition. 72 patients with NYHA functional class (FC) II-III angina pectoris and 40 healthy persons at the age of 47-65 years were examined in a controlled cohort study. Enzyme immunoassay was employed to determine the serum concentrations of interleukins (IL) (IL-2, IL-12, IL-17A, and IL-24), the vasoactive molecules of bradykinin, serotonin, ACE, angiotensin-II (AT-II), NO, and endothelin-1 (ET-1), and plasma renin activity. In addition, the plasma level of the tetrapeptide N-acetyl-Ser-Asp-Lys-Pro was used as a marker for ACE inhibition. The patients with CHD occurring in the presence of hypertension compared with the apparently healthy individuals displayed decreased ET-1 and NO production along with elevated levels of serotonin, AT-II, as well as IL-17A and IL-12. The found changes were accompanied by reduced renin activity. Thus, the individuals with low ACE inhibitor levels showed more pronounced production of the proinflammatory cytokine IL-17A, as well as high plasma concentrations of ACE and NO. The high ACE inhibitor level that reflects patient adherence to appropriate antihypertensive therapy is associated with the reduced production of IL-2 and with the minimum serum levels of ACE, AT-II, and NO, being characterized by the high production of IL-12 and serotonin at the same time. In patients with CHD and hypertension, the high plasma enzyme inhibitor concentration that reflects the activity of appropriate antihypertensive therapy, by contributing to the strengthening of the mechanisms of relaxation of blood vessels, is associated with the risk for proinflammatory activation of whole blood cells and platelets. The mean ACE inhibitor levels that reflect moderate RAAS suppression and are characterized by a relatively low proinflammatory activation of mononuclear cells may be more preferable than the maximum ones, from the point of view of slowing the progression of the subclinical inflammatory process of the vascular wall and preventing possible CHD exacerbations. This determines the feasibility of estimating the plasma level of an ACE inhibitor to control the depth of inhibition of RAAS activity.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

Species specificity of an adrenal androgen-mediated kill-switch triggered by p53 inactivation

Glucose-6-phosphate dehydrogenase (G6PD) is an oncoprotein that is regulated by the p53 tumor suppressor. Mutant p53 loses the ability to inhibit G6PD, and loss of G6PD control clearly plays a role in oncogenesis. The steroid hormone precursor dehydroepiandrosterone (DHEA) is an endogenous uncompetitive inhibitor of G6PD. In humans, and a few other species, the sulfated circulatory form of DHEA (DHEAS) is present at extremely high concentrations – much higher than can be accounted for by DHEA’s function as a precursor to steroid hormones. Uncompetitive inhibition is extremely rare in natural systems because it is irreversible in the presence of high concentrations of substrate and inhibitor. What has gone unappreciated is that such uncompetitive inhibition can quickly lead to cell death when the target is an obligatory housekeeping gene such as G6PD. Cells with inactivated p53 not only lose control over G6PD, but also over hexokinase (HK), the enzyme that converts glucose into glucose-6-phosphate (G6P), the substrate of G6PD. Furthermore, loss of p53 function de-represses NFκB activity, resulting in the upregulation of steroid sulfatase (SS) which converts circulating DHEAS into active DHEA. We propose that inactivation of p53 rapidly elevates G6P and DHEA concentrations in affected cells, driving uncompetitive inhibition of G6PD to lethal irreversibility. In animals with circulating DHEAS, this kill-switch mechanism may prevent most cases of p53 inactivation from becoming tumorigenic. Tumors would thus represent instances in which this mechanism had not been triggered, but which might still be triggered by application of DHEA sufficient to uncompetitively inhibit tumor G6PD. To test this hypothesis, we performed a pilot study in which dogs with cardiac hemangiosarcoma were treated with high dose (HD) DHEA supplemented with isoprene precursors to maintain geranylation of Rac GTPase. Tumor regression and longevity observed in these dogs supported the concept that some tumors retain extraordinary sensitivity to uncompetitive inhibition by DHEA.

Hydrate Equilibrium Modelling with the Cubic two-state Equation of State

A combination of the cubic two-state equation of state and the van der Waals–Platteeuw model was employed for the description of the hydrate formation thermodynamics, with and without inhibitors. The model parameters were determined from the properties of pure compounds and from phase equilibrium data of binary gas–water and water–inhibitor mixtures. With these parameters, predictions were performed for multicomponent gas–water–inhibitor systems. For single-gas hydrates, the average absolute deviations found for equilibrium pressure did not exceed 8%, whereas for multi-gas hydrates, these deviations were 23% maximum. The proposed model produced good predictions in the presence of inhibitors (methanol, ethanol, ethylene glycol and triethylene glycol), with increasing deviations at higher concentrations of inhibitors.

Targeting DNA hypermethylation: Computational modeling of DNA demethylation treatment of acute myeloid leukemia

ABSTRACTIn acute myeloid leukemia (AML) DNA hypermethylation of gene promoters is frequently observed and often correlates with a block of differentiation. Treatment of AML patients with DNA methyltransferase inhibitors results in global hypomethylation of genes and, thereby, can lead to a reactivation of the differentiation capability. Unfortunately, after termination of treatment both hypermethylation and differentiation block return in most cases. Here, we apply, for the first time, a computational model of epigenetic regulation of transcription to: i) provide a mechanistic understanding of the DNA (de-) methylation process in AML and; ii) improve DNA demethylation treatment strategies. By in silico simulation, we analyze promoter hypermethylation scenarios referring to DNMT dysfunction, decreased H3K4me3 and increased H3K27me3 modification activity, and accelerated cell proliferation. We quantify differences between these scenarios with respect to gene repression and activation. Moreover, we compare the scenarios regarding their response to DNMT inhibitor treatment alone and in combination with inhibitors of H3K27me3 histone methyltransferases and of H3K4me3 histone demethylases. We find that the different hypermethylation scenarios respond specifically to therapy, suggesting that failure of remission originates in patient-specific deregulation. We observe that inappropriate demethylation therapy can result even in enforced deregulation. As an example, our results suggest that application of high DNMT inhibitor concentration can induce unwanted global gene activation if hypermethylation originates in increased H3K27me3 modification. Our results underline the importance of a personalized therapy requiring knowledge about the patient-specific mechanism of epigenetic deregulation.

Corrosion Inhibition of Mg Alloy AZ31 By Selenite (SeO32-)

The study of Mg corrosion inhibition by selenium/selenite is very limited. The first relevant work was done by Guy and Whitby in the 1930s1. That work used an acidic selenite-based coating bath to convert Mg alloy surfaces for increased corrosion resistance. It was found that the selenite can react on Mg alloys surfaces to form a protective film. After that report, there was no work reported until Kaminski showed evidence of corrosion inhibition in free corrosion exposure experiments2. The present study follows the work of Kaminski and develops a more comprehensive characterization of Mg corrosion inhibition by selenite using electrochemical and surface analytical methods. Results from these studies support the distinctive qualities of Se films and provide insights on the corrosion inhibition mechanism and inform technological approaches for using this corrosion inhibitor in practical applications. Corrosion inhibition was characterized using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) at three different concentrations of Na2SeO3, which ranged from 1 mM to 50 mM in a base solution of aerated 0.1 M NaCl. After 8 hours immersion in NaCl/inhibitor solution, anodic polarization showed that at low inhibitor concentration the corrosion current density was decreased to less than 10-6 A/cm2. EIS indicated that a low concentration of sodium selenite provided better corrosion protection, as evidenced by an increase in polarization resistance. During these characterizations, a black or red film formed on substrates after immersion tests. A red film was observed at high selenite concentrations, and some trapped transparent crystals were also noted. Several repeat experiments indicated that after three days immersion, nearly all samples were free of obvious corrosion defects. Raman spectroscopy was employed to analyze the surface chemistry. Amorphous selenium tended to form at low inhibitor concentrations, and the film formed was black. Crystallized selenium only formed at high inhibitor concentrations, and the film formed was red. Transparent insoluble crystals were trapped in the red film. Raman spectroscopy showed that these crystals were insoluble MgSeO3. Overall, the corrosion inhibition by selenite is better than that conferred by vandate, but not as good as that conferred by chromate under similar experimental conditions. Among the various film morphologies observed, the amorphous black film provided better corrosion protection than the crystallized red film, as evidenced by electrochemical testing results. In this presentation, details from electrochemical characterization and surface analysis will be discussed in the context of corrosion protection mechanism and prospects for use in a technological application.

Synthesis and corrosion inhibition effects of quinazolin-(3H)-4-one derivatives containing long-chain pyridinium salts on carbon steel in 1.5 M HCl

Quinazolin-4(3H)-one derivatives containing long-chain pyridinium salts were synthesized by a quaternization reaction of pyridine. The confirmation of the synthesized compounds structure was performed by FT-IR, 1H NMR and 13C NMR spectroscopy. Corrosion inhibition tests were performed at room temperature for 24 h on carbon steel coupons in 1.5 M HCl using the weight loss measurements. After the corrosion tests of the compounds it has shown that almost all of the prepared pyridinium salts showed efficient corrosion inhibition at higher inhibitor concentrations (100–250 ppm). Nevertheless, the quinazolin-(3H)-4-one derivative with the longest alkyl chain showed fairly good inhibition in acidic media. To support and establish the corrosion inhibition test results several surface imaging and characterization studies (contact angle, optical profilometer and SEM) were also performed.