An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus—Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and 213 µg/g fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.
Agaricales* ; Fungi ; Humans ; Methods* ; Mycelium ; Parents ; Polymerase Chain Reaction ; Polysaccharides* ; Real-Time Polymerase Chain Reaction ; RNA*

Objective:To explore the effect of the etoposide (VP-16) on the reactivation of human immunodeficiency virus (HIV-1) from latent infection and study its potential molecular mechanism.Methods:The HIV-1 latently infected cell line J-Lat was treated with DMSO, VP-16 and vorinostat (SAHA), flow cytometry was used to detect the positive ratio of green fluorescent protein (GFP) in J-Lat, which can reflect the efficacy of high concentration VP-16 on reactivation. Then the reactivation efficiency of VP-16 on HIV-1 latently infected cells at different concentration and treatment time was measured by flow cytometry. The expression of CD25, CD69 and interleukin-6 (IL-6) of the VP-16-treated J-Lat cells were also detected in the same way. The effect of VP-16 on the transcription of long terminal repeat (LTR) was tested using a dual-luciferase reporter assay. The protein expression of the silent information regulator 1 (SIRT1) and the acetylated nuclear factor κB (NF-κB) p65 under the effect of VP-16 was checked by western blot (WB). Lentivirus-mediated SIRT1 short-hairpin RNA (SIRT1-shRNA) was employed to knock down SIRT1, and then tested for efficiency of reactivation.Results:The results of flow cytometry showed that VP-16 specifically reactivated HIV-1 latently infected J-Lat cells in a concentration and time-dependent manner. Meanwhile, the expression of CD25 and CD69 was upregulated to a certain extent, but the expression of IL-6 was not affected. Dual-luciferase reporter assay suggested that VP-16 positively regulated the transcription of HIV-1 LTR through NF-κB signaling pathway. WB results indicated that VP-16 can inhibit the protein expression of SIRT1 and promote the acetylation of NF-κB p65. Lentivirus-mediated SIRT1-shRNA successfully knocked down the mRNA and protein expression of SIRT1, and reactivated the HIV-1 latency effectively.Conclusions:VP-16 upregulates the acetylation of NF-κB p65 by inhibiting the expression of SIRT1, which subsequently promotes the transcription of HIV-1 LTR and reactivates the latent HIV-1 infection.

Objective To study the quality comparability of human coagulation factor Ⅷ(FⅧ)produced before and after the change of factory site.Methods A comparative study was carried out on quality quantitative indexes,related im-purities and stability data of FⅧ produced before and after the change of factory site.Results The FⅧ quantitative quality before and after the change of factory site all met the quality standard,and the related impurities including aluminum resi-due,tributyl phosphate residue,polysorbate 80 residue and PEG residue all met the quality standard.Other impurities in-cluding human fibrinogen,fibronectin,plasminogen,IgA,IgM and IgG were extremely low in content and equivalent in quality.The content of VWF(von Willebrand factor)had no obvious change before and after the change of factory site,but was significantly higher than that of other domestic manufacturers'commercial products.The results of accelerated stability and long-term stability tests showed that the titer of FⅧ fluctuated within the methodological error range,and the results all met the quality standard.Conclusion The change of factory site of FⅧ has no effect on the quality.