WD40-REPEAT 5a represses root meristem growth by suppressing auxin synthesis through changes of nitric oxide accumulation in Arabidopsis.

Although nitric oxide (NO) is known to regulate root growth, the factor(s) modulating NO during this process have not yet been elucidated. Here, we identified Arabidopsis WD40-REPEAT 5a (WDR5a) as a novel factor that functions in root growth by modulating NO accumulation. The wdr5a-1 mutant accumulated less NO and produced longer roots than the wild type, whereas the WDR5a overexpression lines had the opposite phenotype. The role of NO was further supported by our observation that the NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) rescued the root meristem growth phenotypes of the wdr5a-1 and WDR5a overexpression lines, respectively. The regulation of root growth by WDR5a was found to involve auxin because the auxin levels were similar in SNP-treated wdr5a-1 and wild-type roots, but higher in untreated wdr5a-1 roots than in wild-type roots. In addition, the wdr5a-1 mutant had higher production and activity levels of the auxin biosynthetic enzyme TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1), in contrast to its reduced expression and activity in the WDR5a overexpression lines, and the increased root meristem growth in wdr5a-1 was suppressed by treatment with l-kynurenine, which inhibits TAA1, as well as by mutating TAA1. WDR5a therefore functions in root meristem growth by maintaining NO homeostasis, and thus TAA1-mediated auxin biosynthesis.

CATALASE2 functions for seedling postgerminative growth by scavenging H2 O2 and stimulating ACX2/3 activity in Arabidopsis.

Increased fatty acid ß-oxidation is essential for early postgerminative growth in seedlings, but high levels of H2 O2 produced by ß-oxidation can induce oxidative stress. Whether and how catalase (CAT) functions in fine-tuning H2 O2 homeostasis during seedling growth remain unclear. Here, we report that CAT2 functions in early seedling growth. Compared to the wild type, the cat2-1 mutant, with elevated H2 O2 levels, exhibited reduced root elongation on sucrose (Suc)-free medium, mimicking soils without exogenous sugar supply. Treatment with the H2 O2 scavenger potassium iodide rescued the mutant phenotype of cat2-1. In contrast to the wild type, the cat2-1 mutant was insensitive to the CAT inhibitor 3-amino-1,2,4-triazole in terms of root elongation when grown on Suc-free medium, suggesting that CAT2 modulates early seedling growth by altering H2 O2 accumulation. Furthermore, like cat2-1, the acyl-CoA oxidase (ACX) double mutant acx2-1 acx3-6 showed repressed root elongation, suggesting that CAT2 functions in early seedling growth by regulating ACX activity, as this activity was inhibited in cat2-1. Indeed, decreased ACX activity and short root of cat2-1 seedlings grown on Suc-free medium were rescued by overexpressing ACX3. Together, these findings suggest that CAT2 functions in early seedling growth by scavenging H2 O2 and stimulating ACX2/3 activity.

High-level expression and purification of the major house dust mite allergen Der p 2 in Escherichia coli.

Der p 2, a major allergen derived from the house dust mite Dermatophagoides pteronyssinus, is one of the most clinically relevant allergens worldwide. Recombinant Der p 2 (rDer p 2) is useful in clinical diagnosis and disease-specific immunotherapy. However, previous studies showed that Der p 2 can only be expressed in Escherichia coli (E. coli) cells as inclusion bodies, thus protein refolding is required to obtain functional products. Here we report a new method to produce biologically active Der p 2 protein in E. coli. N-terminal hexahistidine- and trigger factor (TF)-tagged Der p 2 was expressed in soluble form in E. coli and purified using a combination of chromatography processes. This procedure produced milligram-level high purity Der p 2 per liter of bacterial culture. Moreover, far-UV region circular dichroism (CD) analysis and serum specific IgE reactivity test demonstrated that the secondary structure and IgE reactivity properties of rDer p 2 produced in our study were almost identical to those of natural Der p 2 (nDer p 2). In conclusion, the method developed in this work provides a useful tool for the production of immunologically active recombinant Der p 2 for clinical applications.

Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

Development of c­MET­specific chimeric antigen receptor­engineered natural killer cells with cytotoxic effects on human liver cancer HepG2 cells.

In recent years, cellular immunotherapy has served an important role in the combined treatment of hepatocellular carcinoma. The possibility of specific cell therapies for the treatment of solid tumours has been further explored following the success of chimeric antigen receptor (CAR)­T cell therapy in the treatment of haematological tumours. The present study aimed to evaluate the specificity and efficiency of c­MET­targeted CAR­NK cell immunotherapy on human liver cancer in vitro. A CAR structure that targeted and recognised a c­MET antigen was constructed. c­MET­CAR was transferred into primary NK cells using lentiviral infection. c­MET­positive HepG2 cells were used as an in vitro study model. The cytotoxicity assay results revealed that c­MET­CAR­NK cells exhibited more specific cytotoxicity for HepG2 cells with high c­MET expression compared with the lung cancer cell line H1299, which has low levels of c­MET expression. The results of the present study demonstrated that c­MET may be a specific and effective target for human liver cancer cell CAR­NK immunotherapy. Based on these results, CAR­NK cell­based immunotherapy may provide a potential biotherapeutic approach for liver cancer treatment in the future.