Cell survival controlled by lens-derived Sema3A-Nrp1 is vital on caffeine-suppressed corneal innervation during chick organogenesis.

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho-Histone3 (pHIS3) and C-caspase3 revealed that cell survival was repressed by caffeine administration. Whole-mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin-1 (Nrp1) showed that both caffeine and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF-labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A-Nrp1 signaling exerts a key role on the caffeine-suppressed neuron survival. Knocking-down Sema3A through transfection with Sema3A-siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A-Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2-Robo1 and Ephb2. Taken together, we speculate here that caffeine-enhanced reactive oxygen species upregulates Sema3A-Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

[Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui].

According to the previous results from transcriptome analysis of Ligustrum quihoui, a glycosyltransferase gene(xynzUGT) was cloned by rapid amplification of cDNA ends(RACE). The full length cDNA of xynzUGT was 1 598 bp, consisting of 66 bp 5'-UTR, 1 440 bp ORF and 92 bp 3'-UTR. The ORF encoded a 480 amino-acid protein(xynzUGT) with a molecular weight of 54 826.67 Da and isoelectric point of 5.82. The structure of enzyme was analyzed by using bioinformatics method, the results showed that the primary structure contained a highly conserved PSPG box of glycosyltransferase, the secondary structure included α helix(38%), ß sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face α/ß/α domains(often referred to as a Rossmann domains), between which a substrate binding pocket is sandwiched. The phylogenetic tree analysis indicated that xynzUGT might catalyze glycosylation of phenylpropanoids, such as tyrosol. Further simulation experiment of molecular docking between enzyme and tyrosol showed that Gly138 and Ser285 located in the binding pocket interacted with tyrosol by hydrogen bonding. SDS-PAGE analysis exhibited that the prokaryotic expression system successfully expressed recombinant xynzUGT with molecular weight of 58 370.57 Da, but it exists in the form of non-soluble inclusion bodies. Using the molecular chaperone and enzyme co-expression method, the soluble expression was promoted to some extent. The above works laid the foundation for further studying on enzymatic reaction in vitro and clarifying the functional mechanism of enzyme.

De novo transcriptome characterization of Lilium 'Sorbonne' and key enzymes related to the flavonoid biosynthesis.

Lily is an important cut-flower and bulb crop in the commercial market. Here, transcriptome profiling of Lilium 'Sorbonne' was conducted through de novo sequencing based on Illumina platform. This research aims at revealing basic information and data that can be used for applied purposes especially the molecular regulatory information on flower color formation in lily. In total, 36,920,680 short reads which corresponded to 3.32 GB of total nucleotides, were produced through transcriptome sequencing. These reads were assembled into 39,636 Unigenes, of which 30,986 were annotated in Nr, Nt, Swiss-Prot, KEGG, COG, GO databases. Based on the three public protein databases, a total of 32,601 coding sequences were obtained. Meanwhile, 19,242 Unigenes were assigned to 128 KEGG pathways. Those with the greatest representation by unique sequences were for ''metabolic pathways'' (5,406 counts, 28.09 %). Our transcriptome revealed 156 Unigenes that encode key enzymes in the flavonoid biosynthesis pathway including CHS, CHI, F3H, FLS, DFR, etc. MISA software identified 2,762 simple sequence repeats, from which 1,975 primers pairs were designed. Over 2,762 motifs were identified, of which the most frequent was AG/CT (659, 23.86 %), followed by A/T (615, 22.27 %) and CCG/CGG (416, 15.06 %). Based on the results, we believe that the color formation of the Lilium 'Sorbonne' flower was mainly controlled by the flavonoid biosynthesis pathway. Additionally, this research provides initial genetic resources that will be valuable to the lily community for other molecular biology research, and the SSRs will facilitate marker-assisted selection in lily breeding.

Bioinformatic Approach to Investigate Larvae Gut Microbiota Cellulosimicrobium protaetiae via Whole-Genome Analysis.

The insect larvae Protaetia brevitarsis seulensis have recently been researched as a nutritious food source and concentrated on their environmental impacts. Therefore, their gut microbiota has been studied to elucidate their effects and roles on the environment. Of the abundance of bacterial genus identified based on the 16S rRNA genes from isolates of the gut of insect larva Protaetia brevitarsis seulensis, six of the prominent genus were identified as Bacillus (40.2%), Cellulosimicrobium (33.5%), Microbacterium (2.8%), Streptomyces (3%), Krasilnikoviella (17.5%), and Isoptericola (3%) and their similarity of 16S rRNA blast changed from 99 to 100%. Cellulosimicrobium protaetiae BI34T showed strong denitrification and cellulose degradation activity. The newly complete genome sequence of BI34T and the genomes of five species was published in the genus Cellulosimicrobium with emphasis on the denitrification and secondary metabolite genes. In order to elucidate the relationship between the strain BI34T and the host insect larva, the whole-genome sequence was analyzed and compared with the genomes of five strains in the same genus, Cellulosimicrobium, loaded from GenBank. Our results revealed the composition of the gut microbiota of the insect larvae and analyzed the genomic data for the new strain to predict its characteristics and to understand the nitrogen metabolism pathway.

More Liver Metastases Detected Intraoperatively Indicates Worse Prognosis for Colorectal Liver Metastases Patients after Resection Combined with Microwave Ablation.

Background: Whether more tumor numbers detected in surgery compared to preoperative image affecting survival of colorectal liver metastases (CRLM) patients after hepatectomy combined with microwave ablation (MWA) remains unclear. Methods: From 2013 to 2018, 85 CRLM patients who underwent hepatectomy combined with MWA were retrospectively assessed. Compared to the tumor numbers in preoperative image, patients with equal intraoperative tumor numbers were defined as the equal number group (n = 45); patients detected more tumor numbers in surgery were defined as the more number group (n = 40). Clinicopathological factors and prognosis were compared between two groups. Results: Compared to the equal number group, the more number group was characterized by more lymphatic metastasis, synchronous metastasis of liver lesion, and tumor numbers over 5 (all P < 0.05). Median survival time was 46.7 months and 26.8 months in the equal and more number group. Significantly worse overall survival (OS) was found in more number group to the equal number group (P = 0.027). In Cox analysis, more tumor number than image and high level of carbohydrate antigen 19-9 (CA19-9) were poor prognostic factors for OS. Conclusion: In patients receiving hepatectomy combined with MWA, detecting more liver metastases in surgery than preoperative image indicates poor long-term survival. These patients were characterized by more lymphatic metastasis, synchronous metastasis of liver lesion, and tumor numbers over 5. Intensive follow-up to detect early recurrence and potent postoperative therapy to improve survival may be justified in patients detected more tumor numbers in surgery with a high CA19-9 level.

[The condensation mechanism of sodium new houttuyfonate and determination of the chemical structure of condensation products].

To study the condensation mechanism of sodium new houttuyfonate, and determinate the chemical structure of condensation products, dimer was prepared, and LC-DAD-MS/MS multiple techniques were employed to investigate the ultraviolet absorption feature and mass spectrum of transformation solution of dimer, and the transformation kinetics and half-life were studied by ultraviolet spectrophotometry. The pure substance of stable condensation product was obtained by extracting with organic solvent and purifying with column chromatography, the chemical structure of this substance was identified by assaying of IR, HR-ESI-MS and NMR, and the data of LC-MS/MS were compared with that of transformation products of dimer. The results indicated that the dimer is unstable, it will be rapidly dissociated in aqueous solution to form free new houttuyfonate and then cycloaddition reaction will occur and followed by an in situ dehydration to generate 1, 3, 5-tri (dodecanoyl) benzene (trimer) with a six-ring which is stable in aqueous solution. The transformation process may fit second-order kinetics, and the half-times were found to be 3.17 hours at 25 degrees C (298 K) and 6.39 min at 100 degrees C (373 K), separately. It suggests that dimer is an intermediate in condensation reaction, and the end condensation product of sodium new houttuyfonate injection may exist as trimer.