Increasing the heterologous production of spinosad in Streptomyces albus J1074 by regulating biosynthesis of its polyketide skeleton.

Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete, Saccharopolyspora spinosa. However, their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain, and poor adaptability of the heterologous strain to the production of spinosyn. In this study, we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad (spinosyn A and spinosyn D) based on our previous work. We systematically investigated the result of overexpressing polyketide synthase genes (spnA, B, C, D, E) using a constitutive promoter on the spinosad titer in S. albus J1074. The supply of polyketide synthase precursors was then increased to further improve spinosad production. Finally, increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of ∼70 mg/L, which is the highest titer of spinosad achieved in heterologous Streptomyces species. This research provides useful strategies for efficient heterologous production of natural products.

Infusion of gingerols into candied mango enhances shelf-life by inhibiting browning and associated quality parameters during storage.

The study reports shelf-life enhancement of candied mango by infusion of gingerols. Gingerols infused product (GIP), with 3.67 mg gingerols/100 g and non-infused products (control) were packed in multilayer metalized (MET), and ethylene vinyl alcohol (EVOH) based pouches and stored at 25, 35 and 45 °C for 120 days. Degradation kinetics of browning and related parameters showed following order: kß-carotene > ksensory (color) > knon-enzymatic browning > kvitamin C > kantioxidant capacity > k sensory (overall) > ktotal phenolics > kgingerols, resulting in multiple cutoff criteria and predicted shelf-lives (SLpredicted). The application of chemometrics simplified the kinetic interpretations and hence the predictions. Gingerols infusion retarded the deterioration of all quality parameters and substantially enhanced SLpredicted of GIP over control, irrespective of storage conditions. Finally, chemometric based SLpredicted of 144 days closely predicted the actual shelf-life of 142 days for control samples stored in EVOH pouches at 25 °C, in contrast to kinetics based SLpredicted of 185 days.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Identification of galactose-1-phosphate uridyl transferase gene common mutations in dried blood spots.

BACKGROUND: The California newborn screening program uses newborns' dried blood spots (DBS) to screen for more than 45 genetic disorders. Deficiency of galactose-1-phosphate uridyl transferase (GALT) is one of the metabolic genetic disorders screened using newborn DBS. During follow-up tests, common mutations of the GALT gene have been identified using whole blood samples. To avoid the stress of drawing an additional blood sample from newborns who are identified as presumptive positive for galactosemia, we developed a method to test common mutations in the GALT gene using blood spots. METHODS: This method involves DNA extraction from DBS, followed by polymerase chain reaction (PCR), and single nucleotide extension (SNE). SNE products were detected by capillary electrophoresis. RESULTS: In a double-blind study, GALT gene common mutations/variants: IVS2-2A>G, p.S135L, p.T138M, p.Q188R, p.L195P, p.Y209C, p.L218L, p.K285N, and p.N314D were detected in seventy-three DBS which had previously been screened and confirmed as positive in the California Newborn Screening Program. Mutations found using blood spots gave 100% concordance with mutations from previously genotyped whole blood samples. CONCLUSIONS: This blood spot method decreases the genomic test turnaround time of GALT screened positive patients and potentially reduces emotional stress on families required to provide an additional blood draw.

The experimental research anti-tumor effect of modified decoction for driving out blood stasis in the blood mannsion / 国际中医中药杂志

Objective Modified Decoction for Driving Out Blood Stasis in the Blood Mannsion on ascites tumor model of transplanted H22 anti-tumor effect and the impact of VEGF. Methods Adult male mice 100, inoculated with H22 hepatoma cells, the establishment of ascites H22 transplanted tumor model, then divided into 10 groups were given saline, capecitabine, flavored Modified Decoction for Driving Out Blood Stasis in the Blood Mannsion (high, medium and low dose) was administered orally for 10 days, 11 days of treatment, observation of suppression tumor rate, the rate of change in life extension; detected by immunohistochemistry the expression of VEGF in tumors, SPSS13.0 software for statistical analysis. Results Capecitabine, flavored Xuefuzhuyutang (high, medium and low dose) inhibited tumor growth rates were 60%, 49%, 41%, 35%, life extension rate of the three groups were 1.68% 157.98%, 70.58%, 49.57% higher. Conclusion Modified Decoction for Driving Out Blood Stasis in the Blood Mannsion can inhibit tumor cell proliferation in tumor-bearing mice, significantly prolonged the survival time of mice, reduce the tumor tissue and tumor tissue expression of VEGF.