Orthogonal Optimization, Characterization, and In Vitro Anticancer Activity Evaluation of a Hydrogen Peroxide-Responsive and Oxygen-Reserving Nanoemulsion for Hypoxic Tumor Photodynamic Therapy.

Tumor hypoxia can seriously impede the effectiveness of photodynamic therapy (PDT). To address this issue, two approaches, termed in situ oxygen generation and oxygen delivery, were developed. The in situ oxygen generation method uses catalysts such as catalase to decompose excess H2O2 produced by tumors. It offers specificity for tumors, but its effectiveness is limited by the low H2O2 concentration often present in tumors. The oxygen delivery strategy relies on the high oxygen solubility of perfluorocarbon, etc., to transport oxygen. It is effective, but lacks tumor specificity. In an effort to integrate the merits of the two approaches, we designed a multifunctional nanoemulsion system named CCIPN and prepared it using a sonication-phase inversion composition-sonication method with orthogonal optimization. CCIPN included catalase, the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me), photosensitizer IR780, and perfluoropolyether. Perfluoropolyether may reserve the oxygen generated by catalase within the same nanoformulation for PDT. CCIPN contained spherical droplets below 100 nm and showed reasonable cytocompatibility. It presented a stronger ability to generate cytotoxic reactive oxygen species and consequently destroy tumor cells upon light irradiation, in comparison with its counterpart without catalase or perfluoropolyether. This study contributes to the design and preparation of oxygen-supplementing PDT nanomaterials.

An Efficient Strategy Combining Immunoassays and Molecular Identification for the Investigation of Fusarium Infections in Ear Rot of Maize in Guizhou Province, China.

Fusarium is one of the most important phytopathogenic and mycotoxigenic fungi that caused huge losses worldwide due to the decline of crop yield and quality. To systematically investigate the infections of Fusarium species in ear rot of maize in the Guizhou Province of China and analyze its population structure, 175 samples of rotted maize ears from 76 counties were tested by combining immunoassays and molecular identification. Immunoassay based on single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein was first employed to analyze these samples. Fusarium pathogens were isolated and purified from Fusarium-infected samples. Molecular identification was performed using the partial internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) sequences. Specific primers were used to detect toxigenic chemotypes, and verification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS). One-hundred and sixty three samples were characterized to be positive, and the infection rate was 93.14%. Sixteen species of Fusarium belonging to six species complexes were detected and Fusarium meridionale belonging to the Fusarium graminearum species complex (FGSC) was the dominant species. Polymerase chain reaction (PCR) identification illustrated that 69 isolates (56.10%) were potential mycotoxin-producing Fusarium pathogens. The key synthetic genes of NIV, NIV + ZEN, DON + ZEN, and FBs were detected in 3, 35, 7, and 24 isolates, respectively. A total of 86.11% of F. meridionale isolates carried both NIV- and ZEN-specific segments, while Fusarium verticillioides isolates mainly represented FBs chemotype. All the isolates carrying DON-producing fragments were FGSC. These results showed that there are different degrees of Fusarium infections in Guizhou Province and their species and toxigenic genotypes display regional distribution patterns. Therefore, scFv-AP fusion-based immunoassays could be conducted to efficiently investigate Fusarium infections and more attention and measures should be taken for mycotoxin contamination in this region.

First Report of Fusarium miscanthi Causing Ear Rot on Maize in China.

Maize (Zea mays L.), an important food and feed crop worldwide, can be infected by Fusarium pathogens that can contaminate grain with mycotoxins. From August to October in 2018 and 2019, a field survey for maize ear rot was conducted in 76 counties of Guizhou province. The incidence ranged from 3% to 15% at individual fields in different areas. A total of 175 diseased maize ears with similar symptoms, including kernels covered with white, pink or salmon-colored mold or exhibiting a white streaking ("starburst") symptom, were collected from fields. Symptomatic kernels were surface-sterilized by soaking for 30 s in 70% alcohol and for another 2 min in 2% sodium hypochlorite solution, followed by five rinses with sterile water. Each kernel was cut into half and placed on potato dextrose agar (PDA). After incubation at 28 °C in the dark for 5 days, colonies displaying morphological characteristics of Fusarium were transferred to fresh PDA (Leslie and Summerell 2006). Single-sporing was conducted to purify the putative Fusarium colonies. A total of 120 isolates belonged to 16 Fusarium species were determined and F. meridionale was the dominant species. Five isolates from Huaxi district of Guiyang City were identified as F. miscanthi (Gams et al. 1999). Colonies on PDA were white and floccose, and pigmentation as viewed from the underside of the Petri dish was violet. The average growth rate was 7.5-8.0 mm/day at 28 °C in the dark. In cultures grown on PDA, 0-1-septate microconidia were produced in slimy heads. Microconidia were clavate to fusiform with a truncate base and a broadly rounded tip, 4.8-13.3 µm × 1.8-3.3 µm (n=110). In cultures grown on half-strength CMC broth (Xu et al. 2010), macroconidia were mostly 3-septate, almost straight for most of the length, with a slightly foot-shaped basal cell and curved apical cell that gradually tapered, 17.8-71.3 µm × 2.0-4.3 µm (n=78). The identity of the fungus was confirmed by sequence comparison of the partial translation elongation factor-1α (TEF-1α), RNA polymerase II subunit (RPB2), mitochondrial small subunit rDNA (mtSSU) and ß-tubulin genes (Mirete et al. 2004; O'Donnell et al. 2010; O'Donnell and Cigelnik 1997). BLASTn searches of GenBank, using the partial TEF-1α (MN750829), RPB2 (MN750834), mtSSU (MT594104) and ß-tubulin (MT584781) sequences of representative isolate GYHXB03 as the queries, revealed 99.84%, 99%, 100% and 100% sequence identity, respectively, to F. miscanthi NRRL 26231 accessions AF324331, JX171634, AF060371 and AF060384. Inoculum of isolate GYHXB03 was prepared (Xu et al. 2010), and a pathogenicity test was conducted on maize hybrid "Shundan7" and repeated twice. A 106/mL spore suspension (2 mL) or sterile water was injected into each of 8 maize ears through the silk channel at the blister stage of reproductive development in field (Duan et al. 2019). After three weeks, typical Fusarium kernel rot symptoms the same as those previously shown in the field was observed on all pathogen-inoculated plants, while the controls were asymptomatic. The pathogens re-isolated from two diseased kernels were identified as F. miscanthi based on morphology and TEF-1α and mtSSU analyses. F. miscanthi was first isolated from Miscanthus sinensis in Denmark (Gams et al. 1999), and also identified from M. × giganteus rhizomes in Belgium (Scauflaire et al. 2013). To our knowledge, this is the first report of F. miscanthi causing maize ear rot in China. This disease should be monitored in Guizhou due to its threat to maize production.