Group C MAP kinases phosphorylate MBD10 to regulate ABA-induced leaf senescence in Arabidopsis.

Abscisic acid (ABA) is a phytohormone that promotes leaf senescence in response to environmental stress. We previously identified methyl CpG-binding domain 10 (MBD10) as a phosphoprotein that becomes differentially phosphorylated after ABA treatment in Arabidopsis. ABA-induced leaf senescence was delayed in mbd10 knockout plants but accelerated in MBD10-overexpressing plants, suggesting that MBD10 positively regulates ABA-induced leaf senescence. ABA-induced phosphorylation of MBD10 occurs in planta on Thr-89, and our results demonstrated that Thr-89 phosphorylation is essential for MBD10's function in leaf senescence. The in vivo phosphorylation of Thr-89 in MBD10 was significantly downregulated in a quadruple mutant of group C MAPKs (mpk1/2/7/14), and group C MAPKs directly phosphorylated MBD10 in vitro. Furthermore, mpk1/2/7/14 showed a similar phenotype as seen in mbd10 for ABA-induced leaf senescence, suggesting that group C MAPKs are the cognate kinases of MBD10 for Thr-89. Because group C MAPKs have been reported to function downstream of SnRK2s, our results indicate that group C MAPKs and MBD10 constitute a regulatory pathway for ABA-induced leaf senescence.

Accumulation of Phosphorylated SnRK2 Substrate 1 Promotes Drought Escape in Arabidopsis.

Plants adopt optimal tolerance strategies depending on the intensity and duration of stress. Retaining water is a priority under short-term drought conditions, whereas maintaining growth and reproduction processes takes precedence over survival under conditions of prolonged drought. However, the mechanism underlying changes in the stress response depending on the degree of drought is unclear. Here, we report that SNF1-related protein kinase 2 (SnRK2) substrate 1 (SNS1) is involved in this growth regulation under conditions of drought stress. SNS1 is phosphorylated and stabilized by SnRK2 protein kinases reflecting drought conditions. It contributes to the maintenance of growth and promotion of flowering as drought escape by repressing stress-responsive genes and inducing FLOWERING LOCUS T (FT) expression, respectively. SNS1 interacts with the histone methylation reader proteins MORF-related gene 1 (MRG1) and MRG2, and the SNS1-MRG1/2 module cooperatively regulates abscisic acid response. Taken together, these observations suggest that the phosphorylation and accumulation of SNS1 in plants reflect the intensity and duration of stress and can serve as a molecular scale for maintaining growth and adopting optimal drought tolerance strategies under stress conditions.

Evaluation of a deuterated triarylmethyl spin probe for in vivo R2 ∗-based EPR oximetric imaging with enhanced dynamic range.

PURPOSE: In this study, we compared two triarylmethyl (TAM) spin probes, Ox071 and Ox063 for their efficacy in measuring tissue oxygen levels under hypoxic and normoxic conditions by R2 *-based EPR oximetry. METHODS: The R2 * dependencies on the spin probe concentration and oxygen level were calibrated using deoxygenated 1, 2, 5, and 10 mM standard solutions and 2 mM solutions saturated at 0%, 2%, 5%, 10%, and 21% of oxygen. For the hypoxic model, in vivo imaging of a MIA PaCa-2 tumor implanted in the hind leg of a mouse was performed on successive days by R2 *-based EPR oximetry using either Ox071 or Ox063. For the normoxic model, renal imaging of healthy athymic mice was performed using both spin probes. The 3D images were reconstructed by single point imaging and multi-gradient technique was used to determine R2 * maps. RESULTS: The signal intensities of Ox071 were approximately three times greater than that of Ox063 in the entire partial pressure of oxygen (pO2 ) range investigated. The histograms of the tumor pO2 images were skewed for both spin probes, and Ox071 showed more frequency counts at pO2 > 32 mm Hg. In the normoxic kidney model, there was a clear delineation between the high pO2 cortex and the low pO2 medulla regions. The histogram of high-resolution kidney oximetry image using Ox071 was nearly symmetrical and frequency counts were seen up to 55 mm Hg, which were missed in Ox063 imaging. CONCLUSION: As an oximetric probe, Ox071 has clear advantages over Ox063 in terms of sensitivity and the pO2 dynamic range.

Evofosfamide and Gemcitabine Act Synergistically in Pancreatic Cancer Xenografts by Dual Action on Tumor Vasculature and Inhibition of Homologous Recombination DNA Repair.

Aims: Pancreatic ductal adenocarcinomas (PDACs) form hypovascular and hypoxic tumors, which are difficult to treat with current chemotherapy regimens. Gemcitabine (GEM) is often used as a first-line treatment for PDACs but has issues with chemoresistance and penetration in the interior of the tumor. Evofosfamide, a hypoxia-activated prodrug, has been shown to be effective in combination with GEM, although the mechanism of each drug on the other has not been established. We used mouse xenografts from two cell lines (MIA Paca-2 and SU.86.86) with different tumor microenvironmental characteristics to probe the action of each drug on the other. Results: GEM treatment enhanced survival times in mice with SU.86.86 leg xenografts (hazard ratio [HR] = 0.35, p = 0.03) but had no effect on MIA Paca-2 mice (HR = 0.91, 95% confidence interval = 0.37-2.25, p = 0.84). Conversely, evofosfamide did not improve survival times in SU.86.86 mice to a statistically significant degree (HR = 0.57, p = 0.22). Electron paramagnetic resonance imaging showed that oxygenation worsened in MIA Paca-2 tumors when treated with GEM, providing a direct mechanism for the activation of the hypoxia-activated prodrug evofosfamide by GEM. Sublethal amounts of either treatment enhanced the toxicity of other treatment in vitro in SU.86.86 but not in MIA Paca-2. By the biomarker γH2AX, combination treatment increased the number of double-stranded DNA lesions in vitro for SU.86.86 but not MIA Paca-2. Innovation and Conclusion: The synergy between GEM and evofosfamide appears to stem from the dual action of GEMs effect on tumor vasculature and inhibition by GEM of the homologous recombination DNA repair process. Antioxid. Redox Signal. 39, 432-444.

A Simple Preparation Method of Gelatin Hydrogels Incorporating Cisplatin for Sustained Release.

The objective of this study was to develop a new preparation method for cisplatin (CDDP)-incorporated gelatin hydrogels without using chemical crosslinking nor a vacuum heating instrument for dehydrothermal crosslinking. By simply mixing CDDP and gelatin, CDDP-crosslinked gelatin hydrogels (CCGH) were prepared. CDDP functions as a crosslinking agent of gelatin to form the gelatin hydrogel. Simultaneously, CDDP is incorporated into the gelatin hydrogel as a controlled release carrier. CDDP's in vitro and in vivo anticancer efficacy after incorporation into CCGH was evaluated. In the in vitro system, the CDDP was released gradually due to CCGH degradation with an initial burst release of approximately 16%. CDDP metal-coordinated with the degraded fragment of gelatin was released from CCGH with maintaining the anticancer activity. After intraperitoneal administration of CCGH, CDDP was detected in the blood circulation while its toxicity was low. Following intraperitoneal administration of CCGH in a murine peritoneal dissemination model of human gastric cancer MKN45-Luc cell line, the survival time was significantly prolonged compared with free CDDP solution. It is concluded that CCGH prepared by the CDDP-based crosslinking of gelatin is an excellent sustained release system of CDDP to achieve superior anticancer effects with minimal side effects compared with free CDDP solution.

Phosphoproteomic Approaches to Evaluate ABA Signaling.

Abscisic acid (ABA) is a major phytohormone that regulates various processes in plants (e.g., seed dormancy/germination, abiotic/biotic stress responses). As protein phosphorylation is involved in the major pathways of ABA signaling, it is necessary to elucidate the phosphosignaling pathway involved in the ABA response. Phosphoproteomics enables determination of the proteins phosphorylated in vivo, and recent studies have applied a comparative phosphoproteomic approach to analyze ABA signaling in plants. For example, ABA-responsive phosphoproteins were identified in barley embryos. Furthermore, a phosphoproteomic approach is useful for screening protein kinase substrates by comparative analysis using kinase knockout mutants. Here, some technical points regarding phosphoproteomic analyses of ABA responses in plants are described.

Intraperitoneal chemotherapy for peritoneal metastases using sustained release formula of cisplatin-incorporated gelatin hydrogel granules.

PURPOSE: We previously reported the effectiveness of gelatin microspheres incorporating cisplatin in a mouse model of peritoneal metastases. In this study, we report our new complete sustained-release formula of gelatin hydrogel granules incorporating cisplatin (GHG-CDDP), which exerted a good anti-tumor effect with less toxicity. METHODS: GHG-CDDP was prepared without organic solvents to enable its future clinical use. The pharmaceutical characterization of GHG-CDDP was performed, and its in vivo degradability was evaluated. The anti-tumor effect was evaluated using a murine peritoneal metastasis model of the human gastric cancer MKN45-Luc cell line. RESULTS: Our new manufacturing process dramatically reduced the initial burst of CDDP release to approximately 2% (wt), while the previous product had a 25-30% initial burst. In intraperitoneal degradation tests, approximately 30% of GHG-CDDP remained in the murine abdominal cavity 7 days after intraperitoneal injection and disappeared within 3 weeks. GHG-CDDP significantly suppressed the in vivo tumor growth (p = 0.02) and prolonged the survival time (p = 0.0012) compared with the control. In contrast, free CDDP did not show a significant therapeutic effect at any dose. Weight loss and hematological toxicity were also significantly ameliorated. CONCLUSIONS: GHG-CDDP is a promising treatment option for peritoneal metastases through the complete sustained-release of CDDP with less systemic toxicity.

Antiadhesion effect of the C17 glycerin ester of isoprenoid-type lipid forming a nonlamellar liquid crystal.

Postoperative adhesion is a relevant clinical problem that causes a variety of clinical complications after abdominal surgery. The objective of this study is to develop a liquid-type antiadhesion agent and evaluate its efficacy in preventing tissue adhesion in a rat peritoneal adhesion model. The liquid-type agent was prepared by submicron-sized emulsification of C17 glycerin ester (C17GE), squalene, pluronic F127, ethanol, and water with a high-pressure homogenizer. The primary component was C17GE, which is an amphiphilic lipid of one isoprenoid-type hydrophobic chain and can form two phases of self-assembly nonlamellar liquid crystals. The C17GE agent consisted of nanoparticles with an internal inverted hexagonal phase when evaluated by small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM). Upon contact with the biological tissue, this agent formed a thin membrane with a bioadhesive property. After this agent was applied to a sidewall injury of rats, it showed a percentage average of adhesion significantly less than that obtained with the Seprafilm® antiadhesion membrane in a rat model. Additionally, the retention of the agent prolonged at the applied site in the peritoneal cavity of rats. In conclusion, the C17GE agent is promising as an antiadhesion material. STATEMENT OF SIGNIFICANCE: Postoperative adhesion remains a common adverse effect. Although various materials have been investigated, there are few products commercially available to prevent adhesion. For the sheet-type agent, it is inconvenient to be applied through small laparotomy, especially in laparoscopic surgery. Additionally, the liquid-type agent currently used requires a complicated procedure to spray at the targeted site. Our liquid-type antiadhesion agent can form liquid crystals and act as a thin membrane-like physical barrier between the peritoneum and tissues to prevent adhesion. Indeed, the antiadhesion agent used in our present study significantly prevents adhesion compared with the antiadhesion membrane most used clinically. Moreover, our agent is highly stable by itself and easy to use in laparoscopic surgery, thus leading to a promising new candidate as an antiadhesion material.