Very long chain fatty acid metabolism is required in acute myeloid leukemia.

Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.

Analytical Method To Detect and Quantify Avocatin B in Hass Avocado Seed and Pulp Matter.

Avocatin B, an avocado-derived compound mixture, was demonstrated recently to possess potent anticancer activity by selectively targeting and eliminating leukemia stem cells. Avocatin B is a mixture of avocadene and avocadyne, two 17-carbon polyhydroxylated fatty alcohols (PFAs), first discovered in avocado seeds; their quantities in avocado pulp are unknown. Analytical methods to detect avocado seed PFAs have utilized NMR spectroscopy and GC-MS; both of these lack quantitative capacity and accuracy. Herein, we report a sensitive LC-MS method for the quantitation of avocadene and avocadyne in avocado seed and pulp. The method has a reliable and linear response range of 0.1-50 µM (0.03-17.2 ng/µL) for both avocadene and avocadyne ( r2 > 0.990) with a lower limit of quantitation (LLOQ) of 0.1 µM. The intra- and interassay accuracy and precision of the quality control (QC) samples at LLOQ showed ≤18.2% percentage error and ≤14.4% coefficient of variation (CV). The intra- and interassay accuracy and precision for QC samples at low and high concentrations were well below 10% error and CV. This method was successfully applied to quantify avocadene and avocadyne in total lipid extracts of Hass avocado pulp and seed matter.

Fluoxetine-induced transactivation of the platelet-derived growth factor type ß receptor reveals a novel heterologous desensitization process.

Many G protein-coupled receptors (GPCRs), including serotonin (5-HT) receptors promote the activity of receptor tyrosine kinases (RTKs) via intracellular signaling pathways in a process termed transactivation. Although transactivation pathways are commonly initiated by a GPCR, a recent report demonstrated that serotonin-selective reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of the platelet-derived growth factor (PDGF) type ß receptor. We show that a 45 min pretreatment of SH-SY5Y cells with the SSRI fluoxetine indeed blocked 5-HT-induced transactivation of the PDGFß receptor. However, upon further examination, we discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGFß receptor via 5-HT2 receptor activation. After 45min, the increase in PDGFß receptor phosphorylation induced by fluoxetine had returned to baseline, but a subsequent transactivating stimulus (5-HT) failed to "re-transactivate" the PDGFß receptor. We further demonstrate that 45min, but not 3h, 5-HT pretreatment blocks dopamine-induced PDGFß receptor transactivation. This did not involve changes in PDGF receptor function, since ligand (PDGF)-induced PDGFß receptor activation was not inhibited by 5-HT pretreatment. To our knowledge this is the first demonstration of the heterologous desensitization of an RTK transactivation pathway and reveals a previously unknown short-term "blackout" period where no additional transactivation signaling is possible.

Genetic Biodiversity and Posttranslational Modifications of Protease Serine Endopeptidase in Different Strains of Sordaria fimicola.

Genetic variations (mutation, crossing over, and recombination) act as a source for the gradual alternation in phenotype along a geographic transect where the environment changes. Posttranslational modifications (PTMs) predicted modifications successfully in different and the same species of living organisms. Protein diversity of living organisms is predicted by PTMs. Environmental stresses change nucleotides to produce alternations in protein structures, and these alternations have been examined through bioinformatics tools. The goal of the current study is to search the diversity of genes and posttranslational modifications of protease serine endopeptidase in various strains of Sordaria fimicola. The S. fimicola's genomic DNA was utilized to magnify the protease serine endopeptidase (SP2) gene; the size of the product was 700 and 1400 base pairs. Neurospora crassa was taken as the reference strain for studying the multiple sequence alignment of the nucleotide sequence. Six polymorphic sites of six strains of S. fimicola with respect to N. crassa were under observation. Different bioinformatics tools, i.e., NetPhos 3.1, NetNES 1.1 Server, YinOYang1.2, and Mod Pred, to search phosphorylation sites, acetylation, nuclear export signals, O-glycosylation, and methylation, respectively, were used to predict PTMs. The findings of the current study were 35 phosphorylation sites on the residues of serine for protease SP2 in SFS and NFS strains of S. fimicola and N. crassa. The current study supported us to get the reality of genes involved in protease production in experimental fungi. Our study examined the genetic biodiversity in six strains of S. fimicola which were caused by stressful environments, and these variations are a strong reason for evolution. In this manuscript, we predicted posttranslational modifications of protease serine endopeptidase in S. fimicola obtained from different sites, for the first time, to see the effect of environmental stress on nucleotides, amino acids, and proteases and to study PTMs by using various bioinformatics tools. This research confirmed the genetic biodiversity and PTMs in six strains of S. fimicola, and the designed primers also provided strong evidence for the presence of protease serine endopeptidase in each strain of S. fimicola.

Novel Mango Ginger Bioactive (2,4,6-Trihydroxy-3,5-diprenyldihydrochalcone) Inhibits Mitochondrial Metabolism in Combination with Avocatin B.

Acute myeloid leukemia (AML) is an aggressive blood cancer with limited effective chemotherapy options and negative patient outcomes. Food-derived molecules such as avocatin B (Avo B), a fatty-acid oxidation (FAO) inhibitor, are promising novel therapeutics. The roots of the Curcuma amada plants have been historically used in traditional medicine, but isolated bioactive compounds have seldom been studied. Here, we report that 2,4,6-trihydroxy-3,5-diprenyldihydrochalcone (M1), a bioactive from C. Amada, possesses novel anticancer activity. This in vitro study investigated the antileukemia properties of M1 and its effects on mitochondrial metabolism. In combination with Avo B, M1 synergistically reduced AML cell line viability and patient-derived clonogenic growth with no effect on normal peripheral blood stem cells. Mechanistically, M1 alone inhibited mitochondria complex I, while the M1/Avo B combination inhibited FAO by 60%, a process essential to the synergy. These results identified a novel food-derived bioactive and its potential as a novel chemotherapeutic for AML.

Glutamine Metabolism Mediates Sensitivity to Respiratory Complex II Inhibition in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematologic malignancy metabolically dependent on oxidative phosphorylation and mitochondrial electron transport chain (ETC) activity. AML cells are distinct from their normal hematopoietic counterparts by this metabolic reprogramming, which presents targets for new selective therapies. Here, metabolic changes in AML cells after ETC impairment are investigated. Genetic knockdown of the ETC complex II (CII) chaperone protein SDHAF1 (succinate dehydrogenase assembly factor 1) suppressed CII activity and delayed AML cell growth in vitro and in vivo. As a result, a novel small molecule that directly binds to the ubiquinone binding site of CII and inhibits its activity was identified. Pharmacologic inhibition of CII induced selective death of AML cells while sparing normal hematopoietic progenitors. Through stable isotope tracing, results show that genetic or pharmacologic inhibition of CII truncates the tricarboxylic acid cycle (TCA) and leads to anaplerotic glutamine metabolism to reestablish the truncated cycle. The inhibition of CII showed divergent fates, as AML cells lacked the metabolic plasticity to adequately utilize glutamine metabolism, resulting in preferential depletion of key TCA metabolites and death; normal cells were unaffected. These findings provide insight into the metabolic mechanisms that underlie AML's selective inhibition of CII. IMPLICATIONS: This work highlights the effects of direct CII inhibition in mediating selective AML cell death and provides insights into glutamine anaplerosis as a metabolic adaptation that can be therapeutically targeted.

Structure-activity relationship of avocadyne.

Avocado consumption is associated with numerous health benefits. Avocadyne is a terminally unsaturated, 17-carbon long acetogenin found almost exclusively in avocados with noted anti-leukemia and anti-viral properties. In this study, specific structural features such as the terminal triple bond, odd number of carbons, and stereochemistry are shown to be critical to its ability to suppress mitochondrial fatty acid oxidation and impart selective activity in vitro and in vivo. Together, this is the first study to conduct a structure-activity analysis on avocadyne and outline the chemical moieties critical to fatty acid oxidation suppression.

Experience of extracorporeal shockwave lithotripsy for kidney and upper ureteric stones by electromagnetic lithotriptor.

BACKGROUND: Extracorporeal Shock Wave Lithotripsy (ESWL) is a non-invasive treatment of urinary stones which breaks them, by using externally applied, focused, high intensity acoustic pulse, into smaller pieces so that they can pass easily through ureter. Shock wave generation, focusing, coupling and stone localisation by fluoroscope or ultrasound are the basic components of ESWL. ESWL has some complications and is contraindicated in certain situations. The aim of this study was to evaluate the effectiveness and safety of ESWL in kidney and upper ureteric stones by Electromagnetic Lithotriptor. METHODS: All adult patients with renal and upper ureteric stones having a diameter of up to 1 Cm were included in the study. Basic evaluation such as history, examination, ultrasound and excretory urography were performed. Electromagnetic lithotripsy was done and data were collected on a printed proforma from 1st January 2008 to 30th March 2009 in Institute of Kidney Diseases, Peshawar. RESULTS: Out of a total of 625 patients 463 were male and 162 were female; 67.36% of patients were having renal stones, 23.84% upper ureteric and 8.8% both renal and ureteric stones. Complications noted were renal colic in 9.76%, haematuria in 3.2%, steinstrasse in 2.72%, and fever in 1.12% of patients. The stone free rate was 89% and 7% of patients were having stone fragments <4 mm. ESWL failed in 4% of patients. CONCLUSION: ESWL is a safe and effective way of treating kidney and upper ureteric stones.

Avocado-derived polyols for use as novel co-surfactants in low energy self-emulsifying microemulsions.

Avocado (Persea americana Mill.; Lauraceae) seed-derived polyhydroxylated fatty alcohols (PFAs) or polyols (i.e., avocadene and avocadyne) are metabolic modulators that selectively induce apoptosis of leukemia stem cells and reverse pathologies associated with diet-induced obesity. Delivery systems containing avocado polyols have not been described. Herein, natural surface active properties of these polyols are characterized and incorporated into self-emulsifying drug delivery systems (SEDDS) that rely on molecular self-assembly to form fine, transparent, oil-in-water (O/W) microemulsions as small as 20 nanometers in diameter. Mechanistically, a 1:1 molar ratio of avocadene and avocadyne (i.e., avocatin B or AVO was shown to be a eutectic mixture which can be employed as a novel, bioactive, co-surfactant that significantly reduces droplet size of medium-chain triglyceride O/W emulsions stabilized with polysorbate 80. In vitro cytotoxicity of avocado polyol-SEDDS in acute myeloid leukemia cell lines indicated significant increases in potency and bioactivity compared to conventional cell culture delivery systems. A pilot pharmacokinetic evaluation of AVO SEDDS in C57BL/6J mice revealed appreciable accumulation in whole blood and biodistribution in key target tissues. Lastly, incorporation of AVO in SEDDS significantly improved encapsulation of the poorly water-soluble drugs naproxen and curcumin.

Avocatin B Protects Against Lipotoxicity and Improves Insulin Sensitivity in Diet-Induced Obesity.

SCOPE: The effects of an avocado-derived fatty acid oxidation (FAO) inhibitor, avocatin B (AvoB), on glucose and lipid metabolism in models of diet-induced obesity (DIO) and in vitro models of lipotoxicity are evaluated. The safety of its oral consumption in humans is also determined. METHODS AND RESULTS: Mice are given high-fat diets (HFD) for 8 weeks. Thereafter, AvoB or vehicle is administered orally twice weekly for 5 weeks. AvoB inhibits FAO which led to improved glucose tolerance, glucose utilization, and insulin sensitivity. AvoB's effects on metabolism under lipotoxic conditions are evaluated in vitro in pancreatic ß-islet cells and C2C12 myotubes. AvoB inhibits FAO and increases glucose oxidation, resulting in lowering of mitochondrial reactive oxygen species that improves insulin responsiveness in C2C12 myotubes and insulin secretion in INS-1 (832/13) cells, respectively. A randomized, double-blind, placebo-controlled clinical trial in healthy human participants is conducted to assess the safety of AvoB consumption (50 mg or 200 mg per day for 60 days). AvoB is well-tolerated and not associated with any dose-limiting toxicity. CONCLUSION: Therapeutic agents that are safe and effectively inhibit FAO and improve DIO-associated pathologies are currently not available. AvoB's mechanism of action and favorable safety profile highlight its nutritional and clinical importance.

Amyloid-ß Inhibits PDGFß Receptor Activation and Prevents PDGF-BBInduced Neuroprotection.

BACKGROUND: PDGFß receptors and their ligand, PDGF-BB, are upregulated in vivo after neuronal insults such as ischemia. When applied exogenously, PDGF-BB is neuroprotective against excitotoxicity and HIV proteins. OBJECTIVE: Given this growth factor's neuroprotective ability, we sought to determine if PDGF-BB would be neuroprotective against amyloid-ß (1-42), one of the pathological agents associated with Alzheimer's disease (AD). METHODS AND RESULTS: In both primary hippocampal neurons and the human-derived neuroblastoma cell line, SH-SY5Y, amyloid-ß treatment for 24 h decreased surviving cell number in a concentrationdependent manner. Pretreatment with PDGF-BB failed to provide any neuroprotection against amyloid-ß in primary neurons and only very limited protective effects in SH-SY5Y cells. In addition to its neuroprotective action, PDGF promotes cell growth and division in several systems, and the application of PDGFBB alone to serum-starved SH-SY5Y cells resulted in an increase in cell number. Amyloid-ß attenuated the mitogenic effects of PDGF-BB, inhibited PDGF-BB-induced PDGFß receptor phosphorylation, and attenuated the ability of PDGF-BB to protect neurons against NMDA-induced excitotoxicity. Despite the ability of amyloid-ß to inhibit PDGFß receptor activation, immunoprecipitation experiments failed to detect a physical interaction between amyloid-ß and PDGF-BB or the PDGFß receptor. However, G protein-coupled receptor transactivation of the PDGFß receptor (an exclusively intracellular signaling pathway) remained unaffected by the presence of amyloid-ß. CONCLUSIONS: As the PDGF system is upregulated upon neuronal damage, the ability of amyloid-ß to inhibit this endogenous neuroprotective system should be further investigated in the context of AD pathophysiology.

Diffuse reflectance near infrared-chemometric methods development and validation of amoxicillin capsule formulations.

OBJECTIVE: The aim of present study was to establish near infrared-chemometric methods that could be effectively used for quality profiling through identification and quantification of amoxicillin (AMOX) in formulated capsule which were similar to commercial products. In order to evaluate a large number of market products easily and quickly, these methods were modeled. MATERIALS AND METHODS: Thermo Scientific Antaris II near infrared analyzer with TQ Analyst Chemometric Software were used for the development and validation of the identification and quantification models. Several AMOX formulations were composed with four excipients microcrystalline cellulose, magnesium stearate, croscarmellose sodium and colloidal silicon dioxide. Development includes quadratic mixture formulation design, near infrared spectrum acquisition, spectral pretreatment and outlier detection. According to prescribed guidelines by International Conference on Harmonization (ICH) and European Medicine Agency (EMA) developed methods were validated in terms of specificity, accuracy, precision, linearity, and robustness. RESULTS: On diffuse reflectance mode, an identification model based on discriminant analysis was successfully processed with 76 formulations; and same samples were also used for quantitative analysis using partial least square algorithm with four latent variables and 0.9937 correlation of coefficient followed by 2.17% root mean square error of calibration (RMSEC), 2.38% root mean square error of prediction (RMSEP), 2.43% root mean square error of cross-validation (RMSECV). CONCLUSION: Proposed model established a good relationship between the spectral information and AMOX identity as well as content. Resulted values show the performance of the proposed models which offers alternate choice for AMOX capsule evaluation, relative to that of well-established high-performance liquid chromatography method. Ultimately three commercial products were successfully evaluated using developed methods.

5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation.

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

Microbial transformation of 18beta-glycyrrhetinic acid by Cunninghamella elegans and Fusarium lini, and lipoxygenase inhibitory activity of transformed products.

The microbial transformation of 18beta-glycyrrhetinic acid (1) by Cunninghamella elegans afforded a metabolite, 3beta,7beta-dihydroxy-11-oxo-olean-12-en-30-oic acid (2), while fermentation of 1 with Fusarium lini afforded a metabolite, 3,11-dioxo-olean-12-en-30-oic acid (3). Compound 3 exhibited a potent lipoxygenase (LOX) inhibitory activity. The structures of these metabolites were elucidated on the basis of spectroscopic techniques.