Secretory production of 7-dehydrocholesterol by engineered Saccharomyces cerevisiae.

BACKGROUND: 7-Dehydrocholesterol (7-DHC) can be directly converted to vitamin D3 by UV irradiation and de novo synthesis of 7-DHC in engineered Saccharomyces cerevisiae has been recognized as an attractive substitution to traditional chemical synthesis. Introduction of sterol extracellular transport pathway for the secretory production of 7-DHC is a promising approach to achieve higher titer and simplify the downstream purification processing. METHODS AND RESULTS: A series of genes involved in ergosterol pathway were combined reinforced and reengineered in S. cerevisiae. A biphasic fermentation system was introduced and 7-DHC was found to be enriched in oil-phase with an increased titer by 1.5-folds. Quantitative PCR revealed that say1, atf2, pdr5, pry1-3 involved in sterol storage and transport were all significantly induced in sterol overproduced strain. To enhance the secretion capacity, lipid transporters of pathogen-related yeast proteins (Pry), Niemann-Pick disease type C2 (NPC2), ATP-binding cassette (ABC)-family, and their homologues were screened. Both individual and synergetic overexpression of Plant pathogenesis Related protein-1 (Pr-1) and Sterol transport1 (St1) largely increased the de novo biosynthesis and secretory productivity of 7-DHC, and the final titer reached 28.2 mg g-1 with a secretion ratio of 41.4%, which was 26.5-folds higher than the original strain. In addition, the cooperation between Pr-1 and St1 in sterol transport was further confirmed by confocal microscopy, molecular docking, and directed site-mutation. CONCLUSION: Selective secretion of different sterol intermediates was characterized in sterol over-produced strain and the extracellular export of 7-DHC developed in present study significantly improved the cell biosynthetic capacity, which offered a novel modification idea for 7-DHC de novo biosynthesis by S. cerevisiae cell factory.

Phenanthridine Derivative Host Heat Shock Cognate 70 Down-Regulators as Porcine Epidemic Diarrhea Virus Inhibitors.

Porcine epidemic diarrhea virus (PEDV) has become increasingly problematic around the world, not only for its hazards to livestock but also due to the possibility that it is a zoonotic disease. Although vaccine therapy has made some progress toward PEDV control, additional effective therapeutic strategies against PEDV are needed, such as the development of chemotherapeutic agents. The aim of this work was to identify novel anti-PEDV agents by designing and synthesizing a series of phenanthridine derivatives. Among them, three compounds (compounds 1, 2, and 4) were identified as potent anti-PEDV agents exhibiting suppression of host cell heat shock cognate 70 (Hsc70) expression. Mechanism studies revealed that host Hsc70 is involved in the replication of PEDV, and its expression can be suppressed by destabilization of the mRNA, resulting in inhibition of PEDV replication. Activity against PEDV in vivo in PEDV-infected piglets suggested that phenanthridine derivatives are the first host-acting potential anti-PEDV agents.

ITRAQ-Based Proteomics Analysis Reveals the Effect of Neoliensinine on KCl-Induced Vascular Smooth Muscle Contraction by Inhibiting Regulatory Light Chain Phosphorylation.

Smooth muscle (SM) contraction is one of the important physiological functions of the human body, and SM abnormal contraction will induce many diseases. The phosphorylated regulatory light chains (p-RLC) play a decisive role in SM contraction, and dephosphorylation of p-RLC is an effective way to relax SM. Our previous study showed that the novel benzylisoquinoline alkaloid, neoliensinine (Neo), could relax microvascular SM contracted by KCl hyperpolarization. In this study, mesenteric capillaries isolated from 45 mice were divided into normal tension group (Control), 124 mM KCl induced contraction model group (Model), and KCl and Neo-treatment group (Drug). The dephosphorylation levels of RLC in the three groups were measured. Compared with the model group, the phosphorylation of RLC in the drug group was decreased dramatically as expected, suggesting that the relaxation effect of Neo was caused by downregulating p-RLC of microvessel SM. In order to fully understand its fundamental mechanism, our research focused on the identification of target proteins in mice with KCl-induced contractile mesenteric capillary. Isobaric tags for relative and absolute quantification (ITRAQ) tagging was carried out by nanospray liquid chromatography-tandem mass spectrometry. The results allowed the upregulation of 164 differential abundance proteins (DAPs) among the 3,474 protein abundance disturbances identified from the model/control samples. Further comparison showed that there were 16 DAP convergences associated with vascular SM contraction between the drug/model and the drug/control samples. Among them, two proteins with known function, PLCß and RhoGEF12, were selected as target proteins of the relaxation effect of Neo. The two selective target DAPs were verified by Western blot at protein level. The results suggested that changes of the two proteins were consistent with that of the iTRAQ results. Our present work reveals that Neo relaxes vascular smooth muscle via inhibition of RLC phosphorylation, and PLCß and RhoGEF12 may be potential biomarkers for evaluating the effects mediated by Neo.

Genome sequence characterization of canine parvoviruses prevalent in the Sichuan province of China.

Canine parvovirus 2 (CPV-2) infection is responsible for large numbers of animal deaths worldwide and is one of the most dangerous infectious diseases in young puppies. Twenty-four rectal swabs were collected from dogs with clinical signs of vomiting and haemorrhagic diarrhoea and were initially verified to be infected with CPV-2 using colloidal gold test strips. From the 24 CPV-positive samples, complete genome of 5050-5054 nucleotides was sequenced with a next-generation sequencing platform. Characteristics of the Open Reading Frames from different CPV-2 strains detected in this study were analyzed. Several VP2 point mutations were discovered, and demonstrated the co-circulation of new CPV-2a, new CPV-2b and CPV-2c in Sichuan province of China. The analysis results of the Chinese CPV-2 retrieved from the NCBI nucleotide, showed that new CPV-2a has become the predominant variant in some provinces of China. Phylogenetic analysis of global VP2 and NS1 nucleotide sequences revealed certain correlations among geographical regions, types and circulating time, which lays the foundation for further research concerning the epidemiology, genetic variation, vaccination and molecular evolutionary relationships of the CPV-2 identified at different times and from different regions.

Pharmacokinetic Comparisons of Typical Constituents in Curcumae Rhizoma and Vinegar-Processed Curcumae Rhizoma after Oral Administration to Rats.

The Raw Curcumae Rhizoma (R-CR), included in the Chinese Pharmacopoeia Edition 2015, is a well-known Chinese herbal medicine. However, the vinegar-processed Curcumae Rhizoma (V-CR) is used more widely than R-CR. The pharmacokinetics comparison of R-CR and V-CR after oral administration to rats is poorly understood. A novel method, rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-MS) coupled with a sensitive, specific, and convenient microdialysis sampling method, free from endogenous interference was developed in this research. The extracts of R-CR and V-CR were administered orally to each group of rats. The blood and liver microdialysis probes were positioned within the jugular vein toward the right atrium and the median lobe near the center of the liver, respectively. Then, a double-peak phenomenon was observed in the concentration-time curves of curdione in R-CR group, while it was not observed in V-CR group. The liver-to-blood distribution ratio of curdione in V-CR group increased significantly (P < 0.05) compared to that of R-CR group. However, compared with V-CR group, the pharmacokinetic parameters of curcumol exhibited no statistically significant differences from those of R-CR group. These results indicate that vinegar-processed procedure has influence on the pharmacokinetic process of Curcumae Rhizoma in/ns. RRLC-MS coupled with microdialysis system could be used to evaluate the pharmacokinetics of typical constituents in Curcumae Rhizoma after oral administration.

Danggui Buxue Extract-Loaded Liposomes in Thermosensitive Gel Enhance In Vivo Dermal Wound Healing via Activation of the VEGF/PI3K/Akt and TGF-ß/Smads Signaling Pathway.

Danggui Buxue extract-loaded liposomes in thermosensitive gel (DBLTG) are a sustained-release local drug delivery system derived from Danggui Buxue decoction, a well-known Chinese herb formula with wound healing potential. In the present study, we investigated the therapeutic effects of DBLTG on dorsal full-thickness excisional wounds in rats by measuring the percentage of wound contraction and hydroxyproline content, as well as conducting histological observations and immunohistochemical analysis. We also assessed involvement of the vascular endothelial growth factor (VEGF)/phosphatidylinositol 3-kinase (PI3K)/Akt and transforming growth factor beta (TGF-ß)/Smads signaling pathways in the wound healing process upon DBLTG treatment via western blot. The results show that DBLTG treatment remarkably accelerates wound closure, enhances hydroxyproline content in wound granulation tissue, promotes cutaneous wound healing by reducing the inflammatory response and improving fresh granulation tissue formation, and significantly increases the density of blood vessels, cells proliferation, and expression of type I and type III collagen. Moreover, DBLTG markedly upregulates the relative protein expression of VEGFA and TGF-ß1 and notably stimulates the phosphorylation of Akt and Smad2/3. In conclusion, DBLTG significantly improved dermal wound healing in rats by stimulating angiogenesis and collagen synthesis; these effects are likely mediated via the VEGF/PI3K/Akt and TGF-ß/Smads signaling pathways, respectively.

[Effect of vinegar-processed Curcumae Rhizoma on bile metabolism in rats].

To explore the effect of vinegar-processed Curcumae Rhizoma on endogenous metabolites in bile by investigating the endogenous metabolites difference in bile before and after Curcumae Rhizoma was processed with vinegar. Alcohol extracts of crude and vinegar-processed Curcumae Rhizoma, as well as normal saline were prepared respectively, which were then given to the rats by intragastric administration for 0.5 h. Then common bile duct intubation drainage was conducted to collect 12 h bile of the rats. UPLC-TOF-MS analysis of bile samples was applied after 1∶3 acetonitrile protein precipitation; unidimensional statistics were combined with multivariate statistics and PeakView software was compared with network database to identify the potential biomarkers. Vinegar-processed Curcumae Rhizoma extracts had significant effects on metabolites spectrum in bile of the rats. With the boundaries of P<0.05, 13 metabolites with significant differences were found in bile of crude and vinegar-processed Curcumae Rhizoma groups, and 8 of them were identified when considering the network database. T-test unidimensional statistical analysis was applied between administration groups and blank group to obtain 7 metabolites with significant differences and identify them as potential biomarkers. 6 of the potential biomarkers were up-regulated in vinegar-processed group, which were related to the metabolism regulation of phospholipid metabolism, fat metabolism, bile acid metabolism, and N-acylethanolamine hydrolysis reaction balance, indicating the mechanism of vinegar-processed Curcumae Rhizoma on endogenous metabolites in bile of the rats.

The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.