Inducing Effect of Dihydroartemisinic Acid in the Biosynthesis of Artemisinins with Cultured Cells of Artemisia annua

Artemisinin has been used in the production of “artemisinin combination therapies” for the treatment of malaria. Feeding of precursors has been proven to be one of the most effective methods to enhance artemisinin production in plant cultured cells. At the current paper, the biosynthesis of artemisinin (ART) and its four analogs from dihydroartemisinic acid (DHAA) in suspension-cultured cells of Artemisia annua were investigated. ARTs were detected by HPLC/GC-MS and isolated by various chromatography methods. The structures of four DHAA metabolites, namely, dihydro-epi-deoxyarteannuin B, arteannuin I, arteannuin K, and 3-β-hydroxy-dihydro-epi-deoxyarteannuin B, were elucidated by physicochemical and spectroscopic analyses. The correlation between gene expression and ART content was investigated. The results of RT-PCR showed that DHAA could up-regulate expression of amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene C-12 oxidase gene (CYP71AV1), and farnesyl diphosphate synthase gene (FPS) (3.19-, 7.21-, and 2.04-fold higher than those of control group, resp.), which indicated that biosynthesis processes from DHAA to ART were enzyme-mediated

Simulation and experiment of ultrasonic-assisted grinding process for natural diamond

To improve the surface quality of natural diamond grinding, the ultrasonic vibration was introduced for composite grinding, and an inelastic collision theory model was established to calculate the amplitude of ultrasonic vibration. The effects of ultrasonic amplitude, grinding speed and grinding grain size on the material removal of natural diamond (100) crystal surface were studied, and the optimal process parameters were sought. The results show that the ultrasonic amplitude ranges from 3.1 to 8.7 μm in the direction of easy grinding of natural diamond (110) crystal surface and 2.9 to 9.1 μm in the direction of easy grinding of natural diamond (100) crystal surface. The optimal process combination parameters of natural diamond (100) crystal surface grinding obtained by orthogonal experiment are ultrasonic amplitude of 6.0 μm, grinding disk speed of 2 800 r/min, diamond grinding grain size code of M3/6. The surface roughness Ra of diamond (100) crystal surface after ultrasonic assisted grinding is 16.21 nm, which is 63.83% lower than that of traditional mechanical grinding. Ultrasonic assisted grinding of natural diamonds can achieve better surface quality than traditional mechanical grinding

Combinatorial optimization and spatial remodeling of CYPs to control product profile.

Activating inert substrates is a challenge in nature and synthetic chemistry, but essential for creating functionally active molecules. In this work, we used a combinatorial optimization approach to assemble cytochrome P450 monooxygenases (CYPs) and reductases (CPRs) to achieve a target product profile. By creating 110 CYP-CPR pairs and iteratively screening different pairing libraries, we demonstrated a framework for establishing a CYP network that catalyzes six oxidation reactions at three different positions of a chemical scaffold. Target product titer was improved by remodeling endoplasmic reticulum (ER) size and spatially controlling the CYPs configuration on the ER. Out of 47 potential products that could be synthesized, 86% of the products synthesized by the optimized network was our target compound quillaic acid (QA), the aglycone backbone of many pharmaceutically important saponins, and fermentation achieved QA titer 2.23 g/L

RNA helicase DDX5 enables STAT1 mRNA translation and interferon signaling in hepatitis B virus replicating hepatocytes

International audienceObjective: RNA helicase DDX5 is downregulated during hepatitis B virus (HBV) replication, and poor prognosis HBV-related hepatocellular carcinoma (HCC). The objective of this study is to investigate the role of DDX5 in interferon signaling. We provide evidence of a novel mechanism involving DDX5 that enables translation of transcription factor STAT1 mediating the interferon (IFN) response.Design and Results: Molecular, pharmacologic, and biophysical assays were used together with cellular models of HBV replication, HCC cell lines, and liver tumors. We demonstrate that DDX5 regulates STAT1 mRNA translation by resolving a G-quadruplex (rG4) RNA structure, proximal to the 5' end of STAT1 5'UTR. We employed luciferase reporter assays comparing wild type (WT) vs. mutant (MT) rG4 sequence, rG4-stabilizing compounds, CRISPR/Cas9 editing of the STAT1-rG4 sequence, and circular dichroism determination of the rG4 structure. STAT1-rG4 edited cell lines were resistant to the effect of rG4-stabilizing compounds in response to IFN-α, while HCC cell lines expressing low DDX5 exhibited reduced interferon response. Ribonucleoprotein and electrophoretic mobility assays demonstrated direct and selective binding of RNA helicase-active DDX5 to the WT STAT1-rG4 sequence. Immunohistochemistry of normal liver and liver tumors demonstrated that absence of DDX5 corresponded to absence of STAT1. Significantly, knockdown of DDX5 in HBV infected HepaRG cells reduced the anti-viral effect of IFN-α.Conclusion: RNA helicase DDX5 resolves a G-quadruplex structure in 5'UTR of STAT1 mRNA, enabling STAT1 translation. We propose that DDX5 is a key regulator of the dynamic range of interferon response during innate immunity and adjuvant IFN-α therapy

Complete genomic characteristics and pathogenic analysis of the newly emerged classical swine fever virus in China

Abstract Background Classical swine fever (CSF) is one of the most devastating and highly contagious viral diseases in the world. Since late 2014, outbreaks of a new sub-genotype 2.1d CSF virus (CSFV) had caused substantial economic losses in numbers of C-strain vaccinated swine farms in China. The objective of the present study was to explore the genomic characteristics and pathogenicity of the newly emerged CSFV isolates in China during 2014–2015. Results All the new 8 CSFV isolates belonged to genetic sub-genotype 2.1d. Some genomic variations or deletions were found in the UTRs and E2 of these new isolates. In addition, the pathogenicity of HLJ1 was less than Shimen, suggesting the HLJ1 of sub-genotype 2.1d may be a moderated pathogenic isolate and the C-strain vaccine can supply complete protection. Conclusions The new CSFV isolates with unique genomic characteristics and moderate pathogenicity can be epidemic in many large-scale C-strain vaccinated swine farms. This study provides the information should be merited special attention on establishing prevention and control policies for CSF

Primer sets for the amplification of the ORF3 gene and its truncated fragments.

<p>In the F112-GP3 primers, the forward and reverse primers contain <i>Bam</i>HI and <i>Xho</i>I recognition sites, respectively (underlined). Other oligomeric nucleic acid fragments were annealed directively and be used for amino acids pursue absence, which also contain <i>Bam</i>HI and <i>Xho</i>I recognition sites (underlined). Bold font indicates termination codons.</p><p>Primer sets for the amplification of the ORF3 gene and its truncated fragments.</p

Identification of the mAb 3D7 epitope by Western blot.

<p>Western blot analysis of GST-HuN4-F112/F5-N fusion proteins with the mAb 3D7 (A). Lane 1: ultracentrifugal HuN4-F112; lane 2: GST-HuN4-F112-N; lane 3: GST-HuN4-F5-N; lane 4: GST. Truncated fragments were detected with the mAb 3D7 (B). The mAb 3D7 specifically reacted with N protein fragment NF1-3-2 (amino acids 7–33) after three rounds of truncation. F1B is the fragment (amino acids 10–33) identified previously as not recognized by the mAb 3D7 (data not shown), used here as a negative control.</p