Preparation and biomedical application of a non-polymer coated superparamagnetic nanoparticle

We report the preparation of a non-polymer coated superparamagnetic nanoparticle that is stable and biocompatible both in vitro and in vivo. The non-polymer, betaine, is a natural methylating agent in mammalian liver with active surface property. Upon systemic administration, the nanoparticle has preferential biodistribution in mammalian liver and exhibits good reduction of relaxivity time and negative enhancement for the detection of hepatoma nodules in rats using MRI. Our data demonstrate that the non-polymer coated superparamagnetic nanoparticle should have potential applications in biomedicine.     

Identification and functional analysis of LsMNPV anti-apoptosis genes

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.     

Deferoxamine synergistically enhances iron-mediated AP-1 activation: a showcase of the interplay between extracellular-signal-regulated kinase and tyrosine phosphatase

Deferoxamine (DFO) is a drug widely used for iron overload treatment to reduce body iron burden. In the present study, it was shown in mouse epidermal JB6 cells that all iron compounds transiently induced extracellular signal-regulated kinases (ERK) phosphorylation, whereas DFO further enhanced ERK phosphorylation over long periods. The ERK phosphorylation by DFO treatment appears to be due to the inhibition of MAPK phosphatases (MKP) by DFO. The combined effects of iron-initiated MAPK activation and DFO-mediated MKP inhibition resulted in a synergistic enhancement on AP-1 activities. The results indicate that the interplay between MAPK and MKP is important in regulating the extent of AP-1 activation. It is known that administration of DFO in iron overload patients often results in allergic responses at the injection sites. The results suggest that this synergistic AP-1 activation might play a role in DFO-induced skin immune responses of iron overload patients.     

关于木本石松植物鳞皮木属的正确名称

讨论了木本石松植物鳞皮木属Lepidophloios Stemberg的正确名称。基于Stemberg 1825年的原始描述和相关文献以及模式标本,Lepidophloios实际上是一个晚出的拼写变体,它的原始拼写为Lepidopfloyos。根据《国际植物命名法规》（维也纳法规规则60．1和61．1）,Lepidofloys Stemberg应该是鳞皮木属的正确名称。作者给出了鳞皮木属模式标本的图片和描述。     

beta-sitosterol decreases irradiation-induced thymocyte early damage by regulation of the intracellular redox balance and maintenance of mitochondrial membrane stability

Both radiation injury and oxidation toxicity occur when cells are exposed to ion irradiation (IR), ultimately leading to apoptosis. This study was designed to determine the effect of beta-sitosterol (BSS) on early cellular damage in irradiated thymocytes and a possible mechanism of effect on irradiation-mediated activation of the apoptotic pathways. Thymocytes were irradiated (6 Gy) with or without BSS. Cell apoptosis and apoptosis-related proteins were evaluated. BSS decreased irradiation-induced cell death and nuclear DNA strand breaks while attenuating intracellular reactive oxygen species (ROS) and increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). BSS decreased the release of cytochrome c from mitochondria to the cytosol and the mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, BSS partially inhibited the radiation-induced increase of cleaved caspase 3 and cleaved PARP, and attenuated the activation of JNK and AP-1. In addition, evidence suggests that ROS generated by irradiation are involved in this course of cell damage. The results indicate that BSS confers a radioprotective effect on thymocytes by regulation of the intracellular redox balance which is carried out via the scavenging of ROS and maintenance of mitochondrial membrane stability.     

Essential dynamics sampling study of adenylate kinase: comparison to citrate synthase and implication for the hinge and shear mechanisms of domain motions

Essential dynamics sampling simulations of the domain conformations of unliganded Escherichia coli adenylate kinase have been performed to determine whether the ligand-induced closed-domain conformation is accessible to the open unliganded enzyme. Adenylate kinase is a three- domain protein with a central CORE domain and twoflanking domains, the LID and the NMPbind domains. The sampling simulations were applied to the CORE and NMPbind domain pair and the CORE and LID domain pair separately. One aim is to compare the results to those of a similar study on the enzyme citrate synthase to determine whether a similar domain-locking mechanism operates in adenylate kinase. Although for adenylate kinase the simulations suggest that the closed-domain conformation of the unliganded enzyme is at a slightly higher free energy than the open for both domain pairs, the results are radically different to those found for citrate synthase. In adenylate kinase the targeted domain conformations could always be achieved, whereas this was not the case in citrate synthase due to an apparent free-energy barrier between the open and closed conformations. Adenylate kinase has been classified as a protein that undergoes closure through a hinge mechanism, whereas citrate synthase has been assigned to the shear mechanism. This was quantified here in terms of the change in the number of interdomain contacting atoms upon closure which showed a considerable increase in adenylate kinase. For citrate synthase this number remained largely the same, suggesting that the domain faces slide over each other during closure. This suggests that shear and hinge mechanisms of domain closure may relate to the existence or absence of an appreciable barrier to closure for the unliganded protein, as the latter can hinge comparatively freely, whereas the former must follow a more constrained path. In general though it appears a bias toward keeping the unliganded enzyme in the open-domain conformation may be a common feature of domain enzymes.     

Hydrolase-catalyzed Michael addition of 1,3-dicarbonyl compounds to α,β-unsaturated compounds in organic solvent

A novel strategy to perform Michael additions between 1,3-dicarbonyl compounds and α,β-unsaturated compounds was developed by the catalysis of hydrolase. We found that 11 hydrolase could catalyze the enzymatic Michael addition reaction to form the carbon–carbon bond. In 2-methyl-2-butanol d-aminoacylase showed high Michael addition activity. The influence of substrate and Michael acceptor structure on Michael addition was evaluated systematically. Some control experiments demonstrated that the active site of d-aminoacylase was responsible for the enzymatic Michael addition reaction. This novel Michael addition activity of hydrolase is of practical significance in expanding the application of enzymes and in the evolution of new biocatalysts.     

CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry

To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T₀) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.

     

Difference Analysis of ClCYC2-Like Genes Expression and DNA Methylation Between the Two Types of Florets in Chrysanthemum lavandulifolium

DNA methylation is an important epigenetic modification, that is involved in the regulation of gene expression and cell differentiation, and plays an important regulatory role in flower development in higher plants. There are two types of florets on the capitulum in the genus Chrysanthemum, the flower symmetry factor CYCLOIDEA (CYC) 2-like genes may be important candidate genes for determining the identity of the two types of florets. In this study, the diploid plant Chrysanthemum lavandulifolium was used as the research material, and qRT-PCR and bisulfite sequencing polymerase chain reaction (BSP) were used to identify the expression and DNA methylation pattern of CYC2-like genes in the two types of florets. Gene expression analysis showed that the six ClCYC2-like genes were significantly different in the two types of florets, and the expression levels of ClCYC2c, ClCYC2d, ClCYC2e and ClCYC2f in the ray florets were significantly higher than those in the disc florets. For the DNA methylation analysis of the three genes ClCYC2c, ClCYC2d, and ClCYC2e, it was found that the DNA methylation levels of these three genes were negative correlated with their expression levels, and the ways in which the three genes were regulated by the DNA methylation were different. It is speculated that the different DNA methylation of ClCYC2-like genes in the two types of florets may affect the differentiation and development of the two types of florets. This study provides new clues about epigenetics for the analysis of capitulum formation in Asteraceae.