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 plas- mid, viral protein （HBV surface antigen and HBV e an- tigen） secretion was detected by enzyme-linked immu- nosorbent 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-KB （NF-KB） activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Tran- swell 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 re- duced 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-KB signaling was essential for DAI to elicit antivi- ral response in Huh7 cells. When the NF-KB signaling pathway was blocked by a NF-KB signaling suppressor （I~：B^-SR）, the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was indepen- dent of IRF3 signaling and secreted cytokines. CONCLUSION： This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is asso- ciated with activation of NF-KB 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.

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.

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.

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.