Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

Phase 1 Study of a Sulforaphane-Containing Broccoli Sprout Homogenate for Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy worldwide. Our previous results indicate that the reduced oxidative stress capacity of sickle erythrocytes may be caused by decreased expression of NRF2 (Nuclear factor (erythroid-derived 2)-like 2), an oxidative stress regulator. We found that activation of NRF2 with sulforaphane (SFN) in erythroid progenitors significantly increased the expression of NRF2 targets HMOX1, NQO1, and HBG1 (subunit of fetal hemoglobin) in a dose-dependent manner. Therefore, we hypothesized that NRF2 activation with SFN may offer therapeutic benefits for SCD patients by restoring oxidative capacity and increasing fetal hemoglobin concentration. To test this hypothesis, we performed a Phase 1, open-label, dose-escalation study of SFN, contained in a broccoli sprout homogenate (BSH) that naturally contains SFN, in adults with SCD. The primary and secondary study endpoints were safety and physiological response to NRF2 activation, respectively. We found that BSH was well tolerated, and the few adverse events that occurred during the trial were not likely related to BSH consumption. We observed an increase in the mean relative whole blood mRNA levels for the NRF2 target HMOX1 (p = 0.02) on the last day of BSH treatment, compared to pre-treatment. We also observed a trend toward increased mean relative mRNA levels of the NRF2 target HBG1 (p = 0.10) from baseline to end of treatment, but without significant changes in HbF protein. We conclude that BSH, in the provided doses, is safe in stable SCD patients and may induce changes in gene expression levels. We therefore propose investigation of more potent NRF2 inducers, which may elicit more robust physiological changes and offer clinical benefits to SCD patients.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01715480","term_id":"NCT01715480"}}NCT01715480     

Leveraging GWAS data to identify metabolic pathways and networks involved in maize lipid biosynthesis

Maize (Zea mays mays) oil is a rich source of polyunsaturated fatty acids (FAs) and energy, making it a valuable resource for human food, animal feed, and bio‐energy. Although this trait has been studied via conventional genome‐wide association study (GWAS), the single nucleotide polymorphism (SNP)‐trait associations generated by GWAS may miss the underlying associations when traits are based on many genes, each with small effects that can be overshadowed by genetic background and environmental variation. Detecting these SNPs statistically is also limited by the levels set for false discovery rate. A complementary pathways analysis that emphasizes the cumulative aspects of SNP‐trait associations, rather than just the significance of single SNPs, was performed to understand the balance of lipid metabolism, conversion, and catabolism in this study. This pathway analysis indicated that acyl‐lipid pathways, including biosynthesis of wax esters, sphingolipids, phospholipids and flavonoids, along with FA and triacylglycerol (TAG) biosynthesis, were important for increasing oil and FA content. The allelic variation found among the genes involved in many degradation pathways, and many biosynthesis pathways leading from FAs and carbon partitioning pathways, was critical for determining final FA content, changing FA ratios and, ultimately, to final oil content. The pathways and pathway networks identified in this study, and especially the acyl‐lipid associated pathways identified beyond what had been found with GWAS alone, provide a real opportunity to precisely and efficiently manipulate high‐oil maize genetic improvement.     

A Centipede Toxin Family Defines an Ancient Class of CSαβ Defensins

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Insulin is imprinted in the placenta of the marsupial, Macropus eugenii

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.     

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins-PEX2, PEX10 and PEX12-comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.