Treatment Of Wild-type Research Articles

Carbon monoxide promotes stomatal initiation by regulating the expression of two EPF genes in Arabidopsis cotyledons.

The gaseous molecule carbon monoxide (CO) can freely pass through the cell membrane and participate in signal transduction in the cell to regulate physiological activities in plants. Here, we report that CO has a positive regulatory role in stomatal development. Exogenous CO donor CORM-2 [Tricarbonyldichlororuthenium (II) dimer] treatment resulted in an increase of stomatal index (SI) on the abaxial epidermis of cotyledons in wild-type, which can be reversed by the addition of the CO biosynthesis inhibitor ZnPPIX [Protoporphyrin IX zinc (II)]. Consistent with this result, mutation of the CO biosynthesis gene HY1 resulted in a decrease of SI in hy1-100 plants, while overexpression of HY1 led to an increase of SI. Further investigation revealed that CO acts upstream of SPCH and YDA in the stomatal development pathway, since the loss of function mutants spch-1 and yda-2 were insensitive to CORM-2. The expression of EPF2 was inhibited by CORM-2 treatment in wild type and is lower in hy1 than in wild-type plants. In contrast, the expression of STOMAGEN was promoted by CORM-2 treatment and is higher in HY1-overexpression lines. Loss of function mutants of both epf2 and stomagen are insensitive to CORM-2 treatment. These results indicated that CO positively regulates stomatal initiation and distribution by modulating the expression of EPF2 and STOMAGEN.

Antitumor efficacy of MUC1-derived variable epitope library treatments in a mouse model of breast cancer

The identification of novel targets for cancer immunotherapy and the development of new vaccine immunogens are subjects of permanent interest. MUC1 is an overexpressed antigen found in most tumors, and its overexpression correlates with poor prognosis. Many attempts to direct the immune response against MUC1 in tumor cells have failed, including several clinical trials. We have previously developed an innovative Variable Epitope Library (VEL) vaccine platform that carries massively substituted mutant variants of defined epitopes or epitope regions as an alternative to using wild-type peptide sequences-based immunogens. Here, two murine MUC1-derived epitopes equivalent to the previously tested in cancer immunotherapy human MUC1 regions were used to generate VELs. We observed that vaccination with the 23L VEL immunogens, encompassing the entire signal peptide region of MUC1, reduces the tumor area compared to the wild-type sequence treatment. Contrastingly, vaccination with the MUC1 signal peptide-derived predicted CD8++ T cell epitope-based VEL, 9MUC1spL, showed similar tumor area reduction as the wild-type treatment; however, a decrease in lung metastasis after 9MUC1spL treatment was observed. In addition, vaccination induced a large pool of CD8+ T cells which recognized most variant epitopes from 9MUC1spL. Also, we generated MUC1 variable number tandem repeat (VNTR)-based VELs that reduced the metastatic burden when dendritic cells and M13 recombinant bacteriophages were used as vaccine carriers. Collectively, our data demonstrate the immunogenic and antitumor properties of MUC1 signal peptide- and VNTR-derived VEL immunogens.

Thyroid Hormone Receptor α Controls the Hind Limb Metamorphosis by Regulating Cell Proliferation and Wnt Signaling Pathways in Xenopus tropicalis.

Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.

Glucocorticoid-Induced Leucine Zipper-Mediated TLR2 Downregulation Accounts for Reduced Neutrophil Activity Following Acute DEX Treatment.

Glucocorticoids are the most powerful anti-inflammatory and immunosuppressive pharmacological drugs available, despite their adverse effects. Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced gene that shares several anti-inflammatory properties with glucocorticoids. Although immunosuppressive effects of glucocorticoids on neutrophils remain poorly understood, we previously demonstrated that GILZ suppresses neutrophil activation under glucocorticoid treatment. Here, we sought to explore the regulation of Toll-like receptor 2 (TLR2) by the synthetic glucocorticoid dexamethasone (DEX) on neutrophils and the associated GILZ involvement. Peripheral blood neutrophils were isolated from wild type and GILZ-knock-out (KO) mice. TLR2 was found to be downregulated by the in vivo administration of glucocorticoids in wild type but not in GILZ-KO neutrophils, suggesting the involvement of GILZ in TLR2 downregulation. Accordingly, the TLR2-associated anti-fungal activity of neutrophils was reduced by DEX treatment in wild type but not GILZ-KO neutrophils. Furthermore, GILZ did not interact with NF-κB but was found to bind with STAT5, a pivotal factor in the regulation of TLR2 expression. A similar modulation of TLR2 expression, impaired phagocytosis, and killing activity was observed in circulating human neutrophils treated in vitro with DEX. These results demonstrate that glucocorticoids reduce the ability of neutrophils to respond to infections by downregulating TLR2 via GILZ, thereby reducing critical functions.

The EphA4 Signaling is Anti-catabolic in Synoviocytes but Pro-anabolic in Articular Chondrocytes.

The expression and activation of EphA4 in the various cell types in a knee joint was upregulated upon an intraarticular injury. To determine if EphA4 signaling plays a role in osteoarthritis, we determined whether deficient EphA4 expression (in EphA4 knockout mice) or upregulation of the EphA4 signaling (with the EfnA4-fc treatment) would alter cellular functions of synoviocytes and articular chondrocytes. In synoviocytes, deficient EphA4 expression enhanced, whereas activation of the EphA4 signaling reduced, expression and secretion of key inflammatory cytokines and matrix metalloproteases. Conversely, in articular chondrocytes, activation of the EphA4 signaling upregulated, while deficient EphA4 expression reduced, expression levels of chondrogenic genes (e.g., aggrecan, lubricin, type-2 collagen, and Sox9). EfnA4-fc treatment in wildtype, but not EphA4-deficient, articular chondrocytes promoted the formation and activity of acidic proteoglycan-producing colonies. Activation of the EphA4 signaling in articular chondrocytes upregulated Rac1/2 and downregulated RhoA via enhancing Vav1 and reducing Ephexin1 activation, respectively. However, activation of the EphA4 signaling in synoviocytes suppressed the Vav/Rac signaling while upregulated the Ephexin/Rho signaling. In summary, the EphA4 signaling in synoviocytes is largely of anti-catabolic nature through suppression of the expression of inflammatory cytokines and matrix proteases, but in articular chondrocytes the signaling is pro-anabolic in that it promotes the biosynthesis of articular cartilage. The contrasting action of the EphA4 signaling in synoviocytes as opposing to articular chondrocytes may in part be mediated through the opposite differential effects of the EphA4 signaling on the Vav/Rac signaling and Ephexin/Rho signaling in the two skeletal cell types.

Identification of Genes Associated with Sensitivity to Ultraviolet A (UVA) Irradiation by Transposon Mutagenesis of Vibrio parahaemolyticus

Ultraviolet (UV) irradiation is used to disinfect water and food and can be classified as UVA (detected at wavelengths 320–400 nm), UVB (280–320 nm), and UVC (<280 nm). We developed a method for UVA sterilization of equipment with a UVA-light-emitting diode (LED); however, a high rate of fluence was needed to promote pathogen inactivation. The aim of this study was to identify genes associated with UVA sensitivity with the goal of improving UVA-LED-mediated bactericidal activity. We constructed a transposon-mutant library of Vibrio parahaemolyticus and selected six mutants with high sensitivity to UVA irradiation. Genes associated with this phenotype include F-type H+-transporting ATPases (atp), as well as those involved in general secretion (gsp), and ubiquinone and terpenoid-quinone biosynthesis (ubi). Gene complementation resulted in decreased sensitivity to UVA-LED. The atp mutants had lower intracellular adenosine triphosphate (ATP) concentrations than the wild-type treatment, with 20 mM L-serine resulting in elevated ATP concentrations and decreased sensitivity to UVA-LED. The gsp mutants exhibited high levels of extracellular protein transport and the ubi mutants exhibited significantly different intracellular concentrations of ubiquinone-8. Taken together, our results suggest that the protein products of the atp, gsp, and ubi genes may regulate sensitivity to UVA irradiation.

Does targeted therapy dominate in terms of a higher level of effectiveness for a given cost compared to wild-type treatment? An analysis of cost and outcome of metastatic lung cancer patients in Saudi Arabia.

e19361 Background: This study computed and analyzed aggregate and sub-aggregate healthcare costs of lung cancer care by demographic profiles, resource utilization, clinical conditions, and target therapy. Methods: This is a retrospective chart-review and descriptive analysis of resource use and direct medical cost for lung cancer care had at the Oncology Department, King Abdulaziz Medical City for the period 2016-2018. The cost difference between patients receiving targeted therapy (TT) and those with wild-type tumors and the incremental cost-effectiveness ratio (ICER) was performed. The relationship between aggregate cost and demographic profiles, resource utilization, clinical conditions, and treatment therapies using multivariate regression technique was conducted. Results: A total of 35 patients of lung cancer patients with non-small cell lung cancer were analyzed, of which, 34% received targeted therapy; the median age was 62 years (29-84), 43% were ≥ 65 years, 20% were alive; 74% were in stage IV, 69% had visited ED more than twice, and 69% were admitted for non-ICU-related issues. The mean aggregate cost for all patients was USD 197,300 and it was USD 176,000 for TT patients compared with USD 210,600 for non-TT patients. The mean cost for targeted patients was higher for medication components, which accounts for 87.8% of all the costs. On average, the targeted group had USD 34,600 less cost compared with wild type. At least in terms of non-ICU admissions, both groups had on average two admissions. Thus, in terms of ICER, the targeted therapy dominates as it shows lower average aggregate cost for a given level of effectiveness. While longevity (survival) was associated with a higher cost, having stage IV was associated with lower cost for the targeted group compared with wild-type. Generally, being female, > 65 years of age, non-Saudi, and insurance eligibility were associated with lower costs. Conclusions: TT patients had a lower aggregate average cost in spite of expensive medications treatment. This lower aggregate cost presents an added value to the known advantages of patient survival and quality of life.

Insect-resistant Mechanism of Recombinant Baculovirus AcMNPV-PK2-EGFP against Spodoptera exigua Larvae

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a kind of potential biological insecticide. Ac-PK2 protein encoded by orf123 in AcMNPV genome can inhibit eIF2α family kinases to increase viral adaptation in vivo. In our previous study, it was found that recombinant AcMNPV-PK2-EGFP showed high virulence and anti-insect activity against Sf9 cells and Spodoptera exigua larvae. In this study, we investigated the function of Ac-PK2 protein in the insecticidal activity of AcMNPV and the anti-insect mechanism. AcMNPV-PK2-EGFP up-regulated the expression of Ac-pk2 gene in midgut and nerve cord in infected Spodoptera exigua larvae, compared with these in wild-type treatment group. Transcriptional level of BmK IT and detoxication related genes in epidermis, midgut and nerve cord was detected by quantitative PCR. Phenoloxidase activity assay showed that the insect’s humoral immunity was affected by AcMNPV-PK2-EGFP in a time-effect relationship. Western blot analysis of P53 protein indicated the apoptotic level in the midgut tissue was higher in AcMNPV-BmK IT and AcMNPV-PK2-EGFP co-infeced Spodoptera exigua larvae than that in other treatment groups at 4 h and 8 h postinfection. These results provided a theoretical basis to study the application of AcMNPV-PK2-EGFP as a biological insecticide.

Mechanisms of GLP-1 receptor–independent renoprotective effects of the dipeptidyl peptidase type 4 inhibitor linagliptin in GLP-1 receptor knockout mice with 5/6 nephrectomy

Dipeptidyl peptidase type 4 (DPP-4) inhibitors were reported to have beneficial effects in experimental models of chronic kidney disease. The underlying mechanisms are not completely understood. However, these effects could be mediated via the glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP1R) pathway. Here we investigated the renal effects of the DPP-4 inhibitor linagliptin in Glp1r-/- knock out and wild-type mice with 5/6 nephrectomy (5/6Nx). Mice were allocated to groups: sham+wild type+placebo; 5/6Nx+ wild type+placebo; 5/6Nx+wild type+linagliptin; sham+knock out+placebo; 5/6Nx+knock out+ placebo; 5/6Nx+knock out+linagliptin. 5/6Nx caused the development of renal interstitial fibrosis, significantly increased plasma cystatin C and creatinine levels and suppressed renal gelatinase/collagenase, matrix metalloproteinase-1 and -13 activities; effects counteracted by linagliptin treatment in wildtype and Glp1r-/- mice. Two hundred ninety-eight proteomics signals were differentially regulated in kidneys among the groups, with 150 signals specific to linagliptin treatment as shown by mass spectrometry. Treatment significantly upregulated three peptides derived from collagen alpha-1(I), thymosin β4 and heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) and significantly downregulated one peptide derived from Y box binding protein-1 (YB-1). The proteomics results were further confirmed using western blot and immunofluorescence microscopy. Also, 5/6Nx led to significant up-regulation of renal transforming growth factor-β1 and pSMAD3 expression in wild type mice and linagliptin significantly counteracted this up-regulation in wild type and Glp1r-/- mice. Thus, the renoprotective effects of linagliptin cannot solely be attributed to the GLP-1/GLP1R pathway, highlighting the importance of other signaling pathways (collagen I homeostasis, HNRNPA1, YB-1, thymosin β4 and TGF-β1) influenced by DPP-4 inhibition.

Calcium/calmodulin-dependent protein kinase II regulation of IKs during sustained β-adrenergic receptor stimulation

Sustained β-adrenergic receptor (β-AR) stimulation causes pathophysiological changes during heart failure (HF), including inhibition of the slow component of the delayed rectifier potassium current (IKs). Aberrant calcium handling, including increased activation of calcium/calmodulin-dependent protein kinase II (CaMKII), contributes to arrhythmia development during HF. The purpose of this study was to investigate CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and associated functional implications on IKs. KCNQ1 phosphorylation was assessed using liquid chromatography-tandem mass spectrometry after sustained β-AR stimulation with isoproterenol (ISO). Peptide fragments corresponding to KCNQ1 residues were synthesized to identify CaMKII phosphorylation at the identified sites. Dephosphorylated (alanine) and phosphorylated (aspartic acid) mimics were introduced at identified residues. Whole-cell, voltage-clamp experiments were performed in human endothelial kidney 293 cells coexpressing wild-type or mutant KCNQ1 and KCNE1 (auxiliary subunit) during ISO treatment or lentiviral δCaMKII overexpression. Novel KCNQ1 carboxy-terminal sites were identified with enhanced phosphorylation during sustained β-AR stimulation at T482 and S484. S484 peptides demonstrated the strongest δCaMKII phosphorylation. Sustained β-AR stimulation reduced IKs activation (P = .02 vs control) similar to the phosphorylated mimic (P = .62 vs sustained β-AR). Individual phosphorylated mimics at S484 (P=.04) but not at T482 (P = .17) reduced IKs function. Treatment with CN21 (CaMKII inhibitor) reversed the reductions in IKs vs CN21-Alanine control (P < .01). δCaMKII overexpression reduced IKs similar to ISO treatment in wild type (P < .01) but not in the dephosphorylated S484 mimic (P = .99). CaMKII regulates KCNQ1 at S484 during sustained β-AR stimulation to inhibit IKs. The ability of CaMKII to inhibit IKs may contribute to arrhythmogenicity during HF.

Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke.

In response to stroke-induced injury, astrocytes can be activated and form a scar. Inflammation is an essential component for glial scar formation. Previous study has shown that adjudin, a potential Sirt3 activator, could attenuate lipopolysaccharide (LPS)- and stroke-induced neuroinflammation. To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation, we used a transient middle cerebral artery occlusion (tMCAO) model with or without adjudin treatment in wild type (WT) and Sirt3 knockout (KO) mice and performed a wound healing experiment in vitro. Both our in vivo and in vitro results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression. In addition, adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice, which was blunted by Sirt3 deficiency. Furthermore, adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3. Both the suppression of Foxo3a and overexpression of N1ICD could alleviate the inhibitory effect of adjudin in vitro indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing experiment.

The significance of glutathione and ascorbate in modulating the retrograde high light response in Arabidopsis thaliana leaves.

Retrograde signals from the chloroplast control expression of nuclear genes. A large fraction of these genes is affected rapidly upon light intensity shifts. This study was designed to address the interdependence of signaling pathways involved in the rapid high light response and redox and reactive oxygen species signaling by exploiting the glutathione and ascorbate deficient mutants pad2 and vtc1. In the first set of experiments the transcriptional response of the two transcription factors ERF6 and ERF105 that had previously been shown to rapidly respond to light was shown to be deregulated in the pad2 mutant but not in the vtc1 background. The transcriptional response after combining the low-to-high light transfer with methylviologen pretreatment further demonstrated the significance of glutathione in strongly modulating the retrograde response. Transcripts encoding small heat shock proteins (HSP17.4, HSP176a, HSP20-like1 and HSP20-like2) and the lipid transfer protein LTP3 were taken as markers responding to the combinatorial treatment in wild type, and most strongly in pad2 in high light or upon methylviologen treatment. A correlation with H2 O2 accumulation was not observed. It is concluded that glutathione-dependent processes participate in light-triggered rapid gene regulation independent on cellular H2 O2 .

PR-independent neurosteroid regulation of α2-GABA-A receptors in the hippocampus subfields

Progesterone (P) binding to the intracellular progesterone receptors (PRs) plays a key role in epilepsy via modulation of GABA-A receptor plasticity in the brain. This is thought to occur via conversion of P to neurosteroids such as allopregnanolone, an allosteric modulator of GABA-A receptors. In the female brain, the composition of GABA-A receptors is not static and undergoes dynamic spatial changes in response to fluctuations in P and neurosteroid levels. Synaptic α2-containing GABA-A receptors contribute to phasic neuronal excitability and seizure susceptibility. However, the mechanisms underlying α2-subunit plasticity remain unclear. Here, we utilized the neurosteroid synthesis inhibitor finasteride and PR knockout mice to investigate the role of PRs in α2-subunit in the hippocampus. α2-Subunit expression was significantly upregulated during the high-P state of diestrous stage and with P treatment in wildtype and PR knockout mice. In contrast, there was no change in α2-subunit expression when metabolism of P into neurosteroids was blocked by finasteride in both genotypes. These findings suggest that ovarian cycle-related P and neurosteroids regulate α2-GABA-A receptor expression in the hippocampus via a non-PR pathway, which may be relevant to menstrual-cycle related brain conditions.

Cyp1b1-mediated suppression of lymphoid progenitors in bone marrow by polycyclic aromatic hydrocarbons coordinately impacts spleen and thymus: a selective role for the Ah Receptor.

Bone marrow (BM) hematopoietic stem cells differentiate to common lymphoid progenitors (CLP) that emigrate to the thymus to form T cells or differentiate into immature B cells that then migrate to the spleen for maturation. Rapid in vivo suppression of BM progenitor cells by a single oral or intraperitoneal dose of 7,12‐dimethylbenz(a)anthracene (DMBA) subsequently decreased mature lymphoid populations in BM, spleen, and thymus. These suppressions depended on BM CYP1B1, but not on aryl hydrocarbon receptor (AhR) activity. Suppression of pre‐B colony formation at 6 h, correlated with subsequent decreases in mature BM, spleen, and thymus populations (48–168 h). Thymus T‐cell ratios were unaffected, suggesting low local toxicity. DMBA treatment suppressed progenitor cells 24‐h post treatment in wild type (WT), AhRb mice, but not in Cyp1b1‐ko mice. The stem cell populations were sustained. Benzo(a)pyrene (BP) mediated a similar progenitor suppression up to 6 h, but reversal rapidly ensued. This recovery was absent in mice with a polycyclic aromatic hydrocarbon (PAH)‐resistant, AhRd genotype. This AhR‐dependent progenitor recovery with BP induction accounts for the absence of suppression of B220+ BM and spleen populations at 48–168 h. However, DMBA and BP produced similar profiles for thymus cell suppression, independent of AhR genotype. Thus, lymphoid progenitors may exit the BM to the thymus prior to the BP reversal. This progenitor recovery is associated with elevated chemokines and cytokines that depend on AhR‐mediated induction of CYP1A1. This response increased constitutively in Cyp1b1‐ko BM, demonstrating that CYP1B1 metabolizes local stimulants that impact a basal progenitor protection process.

Abstract B21: The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer (PaCa) tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), that are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. We are therefore investigating the role of Prdx-1 and its oxidation states in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data suggests that throughout tumor progression, Prdx-1 is differentially regulated and that its localization changes. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total prdx-1 expression and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by NOX inhibitors and fatty acids that control pancreatic neoplasia. In addition, AKT, ERK , and FOXO3a phosphorylation were also differntially modified by DPI treatment and fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. We therefore conclude that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle A. Schultz, Rital Shah, Brian DeCant, Windel E. Mascarinas, David Bentrem, Paul Grippo. The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B21.

Abstract 3038: G quadruplex stabilization induces mitophagy in S. cerevisiae

Abstract G-quadruplex (G4) DNA structures are stable planar structures held together by multiple G-G base pairs. G4 structures are thought to affect transcription and other chromosomal processes but their presence in vivo is also expected to slow replication fork progression. Previous work from our lab demonstrates that the S. cerevisiae Pif1 5′ to 3′ DNA helicase is a particularly potent unwinder of G4 structures in vitro1. In addition, cells lacking the Pif1 helicase exhibit replication fork slowing and DNA breakage at G4 motifs, chromosomal sequences that form intra-molecular G4 structures in vitro1,2. In addition, treating yeast in vivo with the G4 stabilizing drug PhenDC3 increases G4-mediated genome instability3. We treated a collection of 4,783 S. cerevisiae strains that each contained a single gene deletion with PhenDC3 to search for genes that are sensitive or resistant to PhenDC3. We will discuss results showing that several yeast genes that affect maintenance of mitochondrial DNA and autophagy induction are hypersensitive to PhenDC3. Using fluorescent microscopy and immunoblotting, we further demonstrate that treatment of wild type S. cerevisiae cells with PhenDC3 induces autophagy, and more specifically, mitophagy. Moreover, cells lacking Pif1 are themselves activated for autophagy. Reference: 1 Paeschke, K. et al. Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497, 458-+, doi:10.1038/nature12149 (2013). 2 Paeschke, K., Capra, J. A. &amp; Zakian, V. A. DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678-691, doi:10.1016/j.cell.2011.04.015 (2011). 3 Piazza, A. et al. Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae. Nucleic Acids Res 38, 4337-4348, doi:10.1093/nar/gkq136 (2010). Citation Format: Jennifer Stundon. G quadruplex stabilization induces mitophagy in S. cerevisiae. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3038. doi:10.1158/1538-7445.AM2015-3038

Abstract 2132: Mutant Kras associated protein oxidation in pancreatic neoplasia

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer (PaCa) tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), thioredoxin (Txn), and sulfiredoxin (Srxn), which are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. Txn and Srxn prevent Prdx-1 overoxidation. We are therefore investigating the role of Prdx-1 oxidation and Txn and Srxn regulation in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data suggest that throughout tumor progression, Prdx-1 expression and localization are differentially regulated. These changes also correlated with reduced Txn and Srxn expression in lesions. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total prdx-1 expression and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by NOX inhibitors and omega-3 fatty acids that control pancreatic neoplasia. ERK inhibitors also differentially affected Prdx1 expression in pancreatic cells in vitro. In addition, AKT, ERK, and FOXO3a phosphorylation were also modified by DPI treatment (NOX inhibitor) and omega-3 fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. We therefore conclude that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras-mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle Schultz, Brian DeCant, Andrew Diaz, Sharon Solman, Rital Shah, David Bentrem, Paul J. Grippo. Mutant Kras associated protein oxidation in pancreatic neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2132. doi:10.1158/1538-7445.AM2015-2132

Anti-Aβ antibodies incapable of reducing cerebral Aβ oligomers fail to attenuate spatial reference memory deficits in J20 mice

Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD). A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid β-protein (Aβ), self-associates to form soluble assemblies loosely referred to as “oligomers” and that these are primary mediators of synaptic dysfunction. As such, Aβ, and specifically Aβ oligomers, are targets for disease modifying therapies. Currently, the most advanced experimental treatment for AD relies on the use of anti-Aβ antibodies. In this study, we tested the ability of the monomer-preferring antibody, m266 and a novel aggregate-preferring antibody, 1C22, to attenuate spatial reference memory impairments in J20 mice. Chronic treatment with m266 resulted in a ~70-fold increase in Aβ detected in the bloodstream, and a ~50% increase in water-soluble brain Aβ — and in both cases Aβ was bound to m266. In contrast, 1C22 increased the levels of free Aβ in the bloodstream, and bound to amyloid deposits in J20 brain. However, neither 1C22 nor m266 attenuated the cognitive deficits evident in 12month old J20 mice. Moreover, both antibodies failed to alter the levels of soluble Aβ oligomers in J20 brain. These results suggest that Aβ oligomers may mediate the behavioral deficits seen in J20 mice and highlight the need for the development of aggregate-preferring antibodies that can reach the brain in sufficient levels to neutralize bioactive Aβ oligomers.Aside from the lack of positive effect of m266 and 1C22 on cognition, a substantial number of deaths occurred in m266- and 1C22-immunized J20 mice. These fatalities were specific to anti-Aβ antibodies and to the J20 mouse line since treatment of wild type or PDAPP mice with these antibodies did not cause any deaths. These and other recent results indicate that J20 mice are particularly susceptible to targeting of the APP/Aβ/tau axis. Notwithstanding the specificity of fatalities for J20 mice, it is worrying that the murine precursor (m266) of a lead experimental therapeutic, Solanezumab, did not engage with putatively pathogenic Aβ oligomers.

Abstract A16: Mutant Kras associated protein oxidation in pancreatic neoplasia

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), that are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. We are therefore investigating the role of Prdx-1 and its oxidation states in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data demonstrate that Prdx1 is localized to the nucleus in normal c57-BL6 mice and exclusively located in the cytoplasm in normal pancreatic cells in the EL-Kras model of pancreatic neoplasia. Moreover, in acinar to ductal metaplatic pancreatic cells in EL-Kras mice, there is an increase in cytoplasmic Prdx1 expression and evidence of nuclear staining. Papillary cells in lesions of EL-Kras mice have apical Prdx-1 expression and ductal cells lining these lesions have higher cytoplasmic Prdx-1 expression and some nuclear Prdx-1 localization. This suggests that throughout tumor progression, Prdx-1 is differentially regulated. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total Prdx-1 and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by fatty acids that control pancreatic neoplasia. In addition, ERK phosphorylation was also modified by fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. Our data therefore suggests that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle A. Schultz, Rital Shah, Brian DeCant, David Bentrem, Paul Grippo. Mutant Kras associated protein oxidation in pancreatic neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A16.

Enhanced excision repair and lack of PSII activity contribute to higher UV survival of Chlamydomonas reinhardtii cells in dark

Plant cells are known to differentiate their responses to stress depending up on the light conditions. We observed that UVC sensitive phenotype of light grown asynchronous Chlamydomonas reinhardtii culture (Light culture: LC) can be converted to relatively resistant form by transfer to dark condition (Dark culture: DC) before UVC exposure. The absence of photosystem II (PSII) function, by either atrazine treatment in wild type or in D1 (psbA) null mutant, conferred UV protection even in LC. We provide an indirect support for involvement of reactive oxygen species (ROS) signalling by showing higher UV survival on exposures to mild dose of H2O2 or Methyl Viologen. Circadian trained culture also showed a rhythmic variation in UV sensitivity in response to alternating light–dark (12 h:12 h) entrainment, with maximum UV survival at the end of 12 h dark and minimum at the end of 12 h light. This rhythm failed to maintain in “free running” conditions, making it a non-circadian phenotype. Moreover, atrazine strongly inhibited rhythmic UV sensitivity and conferred a constitutively high resistance, without affecting internal circadian rhythm marker expression. Dampening of UV sensitivity rhythm in Thymine-dimer excision repair mutant (cc-888) suggested the involvement of DNA repair in this phenomenon. DNA excision repair (ER) assays in cell-free extracts revealed that dark incubated cells exhibit higher ER compared to those growing in light, underscoring the role of ER in conferring differential UV sensitivity in dark versus light incubation. We suggest that multiple factors such as ROS changes triggered by differences in PSII activity, concomitant with differential ER efficiency collectively contribute to light–dark (12 h: 12 h) rhythmicity in C. reinhardtii UV sensitivity.