Brassinolide Soaking Reduced Nitrite Content and Extended Color Change and Storage Time of Toona sinensis Bud during Low Temperature and Near Freezing-Point Temperature Storage.

Low temperatures are often used to preserve fruits and vegetables. However, low-temperature storage also causes problems, such as chilling injury, nitrite accumulation, and browning aggravation in plants. This study investigated the effects of brassinolide (BR,1.0 mg L-1) solution soaking, storage temperatures (-2 ± 0.5 °C, 4 ± 0.5 °C, and 20 ± 1 °C), and their combinations on nitrite content, color change, and quality of stored Toona sinensis bud. The results showed that low temperature (LT, 4 ± 0.5 °C) and near freezing-point temperature (NFPT, -2 ± 0.5 °C) storage effectively inhibited the decay of T. sinensis bud compared to room temperature (20 ± 1 °C, the control). The combined treatments of BR with LT or NFPT reduced nitrite content and maintained the color and the contents of vitamin C, carotenoids, saponins, ß-sitosterol, polyphenol, anthocyanin, flavonoids, and alkaloids in T. sinensis bud. BR soaking delayed the occurrence of chilling injury during NFPT storage. Meanwhile, BR soaking enhanced the DPPH radical scavenging activity, ABTS activity, and FRAP content by increasing SOD and POD activity and the contents of proline, soluble, and glutathione, thus decreasing MDA and hydrogen peroxide content and the rate of superoxide radical production in T. sinensis bud during NFPT storage. This study provides a valuable strategy for postharvest T. sinensis bud in LT and NFPT storage. BR soaking extended the shelf life during LT storage and maintained a better appearance and nutritional quality during NFPT storage.

Production of Atosiban's Key Intermediate Pentapeptide: Synthetic Approaches to the Development of a Peptide Synthesis with Less Racemization and Simplifier Purification Process.

The key intermediate NH2-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-COOH of Atosiban was prepared from N-Boc-S-Bzl-cysteine by the stepwise lengthening of the chain according to the repetitive N,O-bis(trimethylsilyl)acetamide/N-hydroxysuccinimide ester (BSA/NHS) strategy. This synthetic route required no chromatography purification and can be readily performed, yielding a highly pure pentapeptide compound.

Melatonin alleviates aluminum-induced growth inhibition by modulating carbon and nitrogen metabolism, and reestablishing redox homeostasis in Zea mays L.

Melatonin, a regulatory molecule, performs pleiotropic functions in plants, including aluminum (Al) stress mitigation. Here, we conducted transcriptomic and physiological analyses to identify metabolic processes associated with the alleviated Al-induced growth inhibition of the melatonin-treated (MT) maize (Zea mays L.) seedlings. Melatonin decreased Al concentration in maize roots and leaves under Al stress. Al stress reduced the total dry weight (DW) by 41.2% after 7 days of treatment. By contrast, the total DW was decreased by only 19.4% in MT plants. According to RNA-Seq, enzyme activity, and metabolite content data, MT plants exhibited a higher level of relatively stable carbon and nitrogen metabolism than non-treated (NT) plants. Under Al stress, MT plants showed higher photosynthetic rate and sucrose content by 29.9% and 20.5% than NT plants, respectively. Similarly, the nitrate reductase activity and protein content of MT plants were 34.0% and 15.0% higher than those of NT plants, respectively. Furthermore, exogenous supply of melatonin mitigated Al-induced oxidative stress. Overall, our results suggest that melatonin alleviates aluminum-induced growth inhibition through modulating carbon and nitrogen metabolism, and reestablishing redox homeostasis in maize. Graphical Abstarct.

Water-Soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as an Effective Gene Delivery Vector.

Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. In this study, a series of polyphosphazenes (PPZ) bearing cyclic polyamine and imidazole groups were synthesized and investigated for gene delivery. Agarose gel electrophoresis assays showed that poly(imidazole/1,4,7,10-tetraazyclodocane)phosphazene (Im-PPZ-cyclen) had good binding ability with plasmid DNA (pDNA), yielding positively charged particles with a size around 120-140 nm from a ratio of 10:1 to 5:1 (Im-PPZ-cyclen/pDNA, w/w). The cytotoxicity of Im-PPZ-cyclen assayed by MTT was lower than that of PEI 25 kDa, and was similar to that reported for poly(di-2-dimethylaminoethylamine)phosphazene (poly(di-DMAEA)phosphazene) to some degree. The maximum transfection efficiency of Im-PPZ-cyclen/pDNA complexes against 293 T cells at the ratio of 5:1 (Im-PPZ-cyclen/pDNA, w/w) is close to that of Lipofectamine 2000. The present work may provide a strategy for the design of new cationic polymers with reduced cytotoxicity and be applied to gene delivery as an efficient nonviral vector.

Cyclen Grafted with poly[(Aspartic acid)-co-Lysine]: Preparation, Assembly with Plasmid DNA, and in Vitro Transfection Studies.

Development of safe and effective gene carriers is the key to the success of gene therapy. Nowadays, it is still required to develop new methods to improve nonviral gene delivery efficiency. Herein, copolymers of poly[(aspartic acid)-co-lysine] grafted with cyclen (cyclen-pAL) were designed and evaluated for efficient gene delivery. Two copolymers with different Asp/Lys block ratios were prepared and characterized by NMR and gel permeation chromatography analysis. Agarose gel retardation, circular dichroism, and fluorescent quenching assays showed the strong DNA-binding and protection ability for the title compounds. Atomic force microscopy studies clearly delineated uniform DNA globules with a diameter around 100 nm, induced by cyclen-pAL. By grafting cyclen on Asp, relatively high gene delivery efficiency and low cytotoxicity of the modified copolymers were achieved compared with their parent compounds. The present work might help to develop strategies for design and modification of polypeptide copolymers, which may also be applied to favorable gene expression and delivery.

An IDB-containing low molecular weight short peptide as an efficient DNA cleavage reagent.

Artificial nucleases have attracted significant interest due to their abilities in accelerating DNA cleavage, which results in the possibility of genome manipulation. However, compared with natural nucleases, the currently available artificial nucleases have low cleavage efficiency, especially metal-free artificial nucleases. Thus, it is still a challenge to develop highly efficient metal-free artificial nucleases via a non-oxidative pathway. We here designed and prepared a group of rigid bis-amine-grafted PASP conjugates (PASP-IDB), and investigated their abilities to induce DNA double-strand cleavage. The detailed assays showed that in the absence of metal ions, these short peptide conjugates can effectively break the phosphodiester linkage at a relatively low concentration and under physiological conditions through a hydrolytic process, giving the 10(7)-fold rate acceleration over uncatalyzed double-strand DNA. The probable mechanism verified by control experiments revealed that IDBs and free carboxyl groups in PASP synergically catalyzed DNA cleavage. In addition, the effects of degrees of substitution on the cleavage activity were studied, and the results indicated the existence of minimum building blocks of PASP-IDB for efficient DNA cleavage. The results of our study have implications on the design of short peptide-based molecules as new artificial nucleases and may provide a strategy for developing safe and efficient metal-free DNA cleavage reagents.

Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought.

Establishing crop cultivars with strong tolerance to P and N deprivation, high salinity, and drought is an effective way to improve crop yield and promote sustainable agriculture worldwide. A vacuolar H+-pyrophosphatase (V-H+-PPase) gene in wheat (TaVP) was functionally characterized in this study. TaVP cDNA is 2586-bp long and encodes a 775-amino-acid polypeptide that contains 10 conserved membrane-spanning domains. Transcription of TaVP was upregulated by inorganic phosphate (Pi) and N deprivation, high salinity, and drought. Transgene analysis revealed that TaVP overexpression improved plant growth under normal conditions and specifically under Pi and N deprivation stresses, high salinity, and drought. The improvement of growth of the transgenic plants was found to be closely related to elevated V-H+-PPase activities in their tonoplasts and enlarged root systems, which possibly resulted from elevated expression of auxin transport-associated genes. TaVP-overexpressing plants showed high dry mass, photosynthetic efficiencies, antioxidant enzyme activities, and P, N, and soluble carbohydrate concentrations under various growth conditions, particularly under the stress conditions. The transcription of phosphate and nitrate transporter genes was not altered in TaVP-overexpressing plants compared with the wild type, suggesting that high P and N concentrations regulated by TaVP were caused by increased root absorption area instead of alteration of Pi and NO3- acquisition kinetics. TaVP is important in the tolerance of multiple stresses and can serve as a useful genetic resource to improve plant P- and N-use efficiencies and to increase tolerance to high salinity and drought.

Overexpression of TaNHX3, a vacuolar Na⁺/H⁺ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes.

Salt stress is one of the major abiotic stresses affecting plant growth, development, and productivity. In this study, we functionally characterized a wheat vacuolar Na(+)/H(+) antiporter gene (TaNHX3). TaNHX3 is 78.9% identical with TaNHX2 in nucleic acid level, encoding a polypeptide of 522 amino acids (aa). TaNHX3 is targeted onto tonoplast after ER sorting and can complement the growth under salt stress in a yeast mutant with a defective vacuolar Na(+)/H(+) antiporter exchange. TaNHX3 transcripts were induced by applying salt stress in wheat cultivars. More TaNHX3 were detected in the salt-stress-resistant cultivar Ji 7369 compared with the salt-stress-sensitive cultivar Shimai 12 and Ji-Shi-3, an isogenic line derived from aforementioned cultivars with Shimai 12 genetic background. The ectopic TaNHX3 expression in tobacco significantly enhanced the plant tolerance to salt stress. Compared with control plants, the TaNHX3 overexpressing plants displayed no varied Na(+) contents and accumulated more Na(+) amount in plants. However, they exhibited higher fresh and dry weights, more accumulative nitrogen, phosphorus, and potassium, higher contents of chlorophyll, carotenoid, soluble protein, higher activities of the antioxidant enzymes including superoxide dismutase, catalase, and peroxidase, and lower malondialdehyde and H2O2 amount. Our results indicated that TaNHX3 plays an important role in regulating the cytosolic Na(+) transportation within vacuoles under high salinity, alleviating the Na(+) damage effects. The improved salt stress tolerance in TaNHX3 overexpressing tobacco plants is closely associated with the improvement of the aforementioned physiological processes. TaNHX3 can be used as a candidate gene for molecular breeding of salt-tolerant plants.

The inhibition of lamellar hydroxyapatite and lamellar magnetic hydroxyapatite on the migration and adhesion of breast cancer cells.

Hydroxyapatite nanoparticles have been reported to exhibit potent anti-tumor effects in some cancer cells. In our previous study, we have successfully synthesized two types of hydroxyapatite nanoparticles, laminated hydroxyapatite (L-HAp) and laminated magnetic hydroxyapatite (LM-HAp). In this study, we wanted to investigate the effects of L-HAp and LM-HAp with various concentrations on human breast cancer MDA-MB-231 cells. Cell proliferation was assessed with a MTT colorimetric assay. Scratch and adhesion assays were used to detect the effects of these two materials on migration and adhesion. The expressions of integrin ß1 and Akt were measured by Western blotting. Our results showed that L-HAp and LM-HAp had little cell cytotoxicity and significantly reduced cell mobility and adhesion. LM-HAp showed greater inhibitor ability on migration and adhesion of MDA-MB-231 cells. Moreover, results from western blotting showed that L-HAp and LM-HAp impacted the phosphorylation of integrin ß1, but showed no regular impact on Akt. This study suggests that L-HAp and LM-HAp may be potential anti-tumor and delivery system for breast cancer therapy.

Molecular cloning and characterization of GuHMGR, an HMG-CoA reductase gene from liquorice (Glycyrrhiza uralensis).

A full length cDNA encoding HMGR (designated as GuHMGR) was isolated from liquorice (Glycyrrhiza uralensis) based on degenerated PCR and genome walking. The full length cDNA of GuHMGR was 2330 bp with a 1518-bp open reading frame (ORF) encoding a 505-aa polypeptide. Bioinformatics analysis indicated that there were two trans-membrane domains in GuHMGR. A molecular model of tertiary structure showed that GuHMGR is a novel HMGR with a similar spatial structure to other plant HMGRs. The deduced polypeptide of GuHMGR has an isoelectric point (pI) of 6.41 and a calculated molecular weight of about 54.7 kDa. Sequence comparison and phylogenetic tree analysis showed that GuHMGR had the highest homology with HMGRs from Pisum sativum and Medicago truncatula, indicating that GuHMGR belongs to the plant HMGR group. Expression analysis showed the similar amount of transcript level of GuHMGR in roots and leaves, suggesting that this gene was expressed constitutively in plants. Therefore, this novel HMGR gene would possibly provide a new strategy for studying the glycyrrhizin metabolism at the molecular level in the future.