DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication

AIM: To investigate whether DNA-dependent activator of interferon-regulatory factors (DAI) inhibits hepatitis B virus (HBV) replication and what the mechanism is. METHODS: After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plasmid, viral protein (HBV surface antigen and HBV e antigen) secretion was detected by enzyme-linked immunosorbent assay, and HBV RNA was analyzed by real-time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 (IRF3) phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-B (NF- B) activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Transwell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines. RESULTS: Viral protein secretion was significantly reduced by 57% (P < 0.05), and the level of total HBV RNA was reduced by 67% (P < 0.05). The viral core particle-associated DNA was also dramatically down-regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-B signaling was essential for DAI to elicit antiviral response in Huh7 cells. When the NF-B signaling pathway was blocked by a NF-B signaling suppressor (I B -SR), the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was independent of IRF3 signaling and secreted cytokines. CONCLUSION: This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is associated with activation of NF-B but independent of IRF3 and secreted cytokines.

Fast Evaluation of Oxidative DNA Damage by Liquid Chromatography-Electrospray Tandem Mass Spectrometry Coupled With Precision-cut Rat Liver Slices

Objective To establish a fast and sensitive method for the detection of 8-hydroxy-2’-deoxyguanosine （8-OHdG） in precision-cut rat liver slices by HPLC-MS/MS and to investigate isoniazid （INH） -induced oxidative DNA damage. Methods Precision-cut liver slices （300 μm） were prepared from male rats, and incubated with INH （0.018 mol/L） for 2 h after 1 h preincubation. DNA in the slices was extracted and digested into free nucleosides at 37℃ . The samples were injected into HPLC-MS/MS after the proteins were removed. The level of oxidative DNA damage was estimated using the ratio of 8-OHdG to deoxyguanosine （dG）. Results The limit of detection of 8-OHdG was 1 ng/mL （S/N=3） and the intra-assay relative standard variation was 3.38% when one transition 284.3/168.4 was used as a quantifier and another two transitions 284.3/140.2, 306.1/190.2 as qualifiers. 8-OHdG and dG were well separated, as indicated by elution at 10.02 and 7.37 min, respectively. INH significantly increased the ratio of 8-OHdG to dG in rat liver slices （P〈0.05）. Conclusion 8-OHdG in precision-cut liver slices could be sensitively determined by HPLC-MS/MS. HPLC-MS/MS coupled with precision-cut tissue slices is a fast and reliable analytical technique to evaluate oxidative DNA damage of target tissues caused by procarcinogens and cytotoxins.

Protective Effects of Hydroxysafflor Yellow A against Oxidative Damage of β-Mercaptoethanol During Neural Differentiation of Mesenchymal Stem Cells

Objective To study the protective effects of hydroxysafflor yellow A （HSYA） against the oxidative damage caused by β-mercaptoethanol （BME） during neural differentiation of mesenchymal stem cells （MSCs） in vitro. Methods When the confluence reached 50%-60%, 4th passage MSCs were divided into three groups to culture. Gt ： normal group which was cultured using basic medium （DMEM containing 10% FBS all the time）; G2： unprotected group which was continuously cultured using basic medium for 24 h, and then cultured using pre-induction medium （DMEM containing 10% FBS and 1 mmol/L BME）; G3： protected group which was firstly cultured using protective medium （DMEM containing 10% FBS and 160 mg/L HSYA） for 24 h, and then cultured using pre-induction medium for 24 h. After these treatments as above, cell viability, relative levels of SOD/GSH and apoptosis rate were respectively detected. The expression of Bcl and Bax was examined by Western blotting. After HSYA protection and BME pre-induction, neural induction was performed. The expression of NSE and MAP-2 was respectively analyzed on cellular and molecular levels. Results Compared with unprotected group, 160 mg/L HSYA could obviously improve cells viability, maintain high level of SOD and GSH in MSCs, reduce apoptosis rate and improve the ratio of Bcl/Bax. After protection with 160 mg/L HSYA, the survival time of neuron-like cells could be extended. Immunocytochemical staining showed that after 10 h of neural induction, the differentiated neuron-like cells in protected group were still in a good state, and the mRNA levels of NSE and MAP-2 were increased during the induction course checked. Conclusion HSYA could improve the resistance of cells to the oxidative damage caused by BME.

Acquired reactive perforating collagenosis

To the Editor:A 50-year-old man presented with a 1-month history of pruritic papules and nodules on his trunk and extremities.The patient had a 20-year history of alcoholism,and a 10-year history of Meniere’s disease.He was treated with intravenous sodium aescinate for 5 days because of Meniere’s disease 2 months ago.Other chronic diseases or special family histories were denied.Upon physical examination,red or brown papules,and nodules with diameter of 3 to 5 mm were noted on his limbs,shoulders,and dorsum,with central umbilicated necrosis,or keratin plug,accompanied by Kobner Phenomenon[Figure 1A].Ultrasonic examination,peripheral blood cell count and biochemical examination were basically normal.Dermoscopic examination revealed a red-brown structureless area covered with crusts and scales centrally,surrounded by a white rim,and a reddish inflammatory circle with looped and dotted vessels peripherally in polarization mode[Figure 1B].Histopathology showed neutrophils and degenerated keratin components in central goblet necrotic epidermis,degenerated collagen fibers beneath the necrotic epidermis,and sheet of lymphocytes and scattered eosinophils around blood vessels in the dermis[Figure 1C].Masson staining[Figure 1D]and Verhoeff-van Gieson staining[Figure 1E]confirmed the penetration of collagen fibers and fragmented elastic fibers in the necrotic epidermis.Acquired reactive perforating collagenosis was diagnosed,which reacted well to 5-week treatment of oral anti-histamines,topical steroids,and narrow-band ultraviolet B.The patient reported clearance of the lesions for 4 months,but recurrence after alcohol intake again in telephone follow-up.

Acceptance of overseas clinical trial data of medical devices for pre-market registration: general principles and considerations of the National Medical Products Administration

A clinical trial is a systematic investigation or study in or on one or more human subjects,undertaken to assess the safety,clinical performance,and/or effectiveness of a medical device.[1]In this paper,“overseas clinical trial data”refers to data generated from a clinical trial(s)conducted in a foreign country or jurisdiction and proposed to be used as clinical evidence for a pre-market registration application in China.