This study aimed to investigate the drug susceptibility of wild-type and mutant avian influenza A (H7N9) virus neuraminidase (NA) to oseltamivir and zanamivir. Codon optimized DNA of H7N9 (A/ Hangzhou/1/2013) NA was synthesized and constructed into the pcDNA3.1/His vector (NA(H7N9-WT)). Mutant NA(H7N9-H274Y) and NA(H7N9-R292K) plasmids were constructed by directed mutagenesis PCR using NA(H7N9-WT) plasmid as the template followed by sequencing. NA plasmids were transfected into 293T cells and cell lysates containing NAs were collected 48 h post-transfection. Wild-type and mutant NAs were analyzed by Western blotting and their activities were tested by the 4-MUNANA-based assay. All three NAs were expressed and enzymatic activities were confirmed. The effects of oseltamivir and zanamivir on all three NAs were then tested. It showed that the half maximal inhibitory concentrations (IC50s) of oseltamivir carboxylate on NA(H7N9-WT), NA(H7N9-H274Y) and NA(H7N9-R292K) were 1.6 nM, 15.1 nM, and > 1 000 nM with fold changes of 9 and > 625, respectively. The IC50 values of zanamivir on NA(H7N9-WT), NA(H7N9-H274Y), and NA(H7N9-R292K) were 1.1 nM, 1.4 nM, and 38.0 nM with fold changes of 1.3 and 34, respectively. These results indicated that oseltamivir and zanamivir could significantly inhibit NA(H7N9-WT). NA(H7N9-R292K) showed high-level resistance to both drugs (34-fold and 625-fold) and NA(H7N9-H274Y) was sensitive to both (1.3-fold and 9-fold). These results indicated that both oseltamivir and zanamivir could be used for patients infected with the H7N9 virus. However, when patients carried the H7N9 virus with a NA R292K mutation, other medications would be preferred over oseltamivir or zanamivir.
Antiviral Agents ; pharmacology ; Humans ; Influenza A Virus, H7N9 Subtype ; drug effects ; enzymology ; genetics ; Influenza, Human ; virology ; Microbial Sensitivity Tests ; Mutation ; Neuraminidase ; antagonists & inhibitors ; genetics ; metabolism ; Oseltamivir ; pharmacology ; Viral Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Zanamivir ; pharmacology

Objective To observe the suppressing effect of topical nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids on ultraviolet ray (UV)-induced erythema.Methods A solar simulator and an UV phototherapy device were used as light sources,respectively.Erythema reaction was induced on the back skin of 30 healthy volunteers by 1,2 and 3 minimal erythema doses (MED) of irradiation.Five preparations including butyl flufenamate 2.5％ ointment,butyl flufenamate 5％ ointment,the base of butyl flufenamate ointment,halometasone ointment,and diclofenac 1％ ointment,were applied to the irradiation sites respectively half an hour before or immediately after the irradiation.One irradiation site remained untreated and served as the control.The degree of erythema was evaluated by a chromameter at 4,24,and 48 hours after the irradiation.Intragroup and intergroup comparisons were done by t test and analysis of variance,respectively.Results When applied half an hour before solar-simulated irradiation,both 2.5％ and 5％ butyl flufenamate ointment totally suppressed the erythema reaction induced by 1-3 MED of UV irradiation,with no significant increase in erythema index at all the three time points after irradiation (all P ＞ 0.05); diclofenac 1％ only inhibited the erythema induced by 1 MED of UV irradiation at 4 and 48 hours,with no difference observed in erythema index between the baseline and these time points after irradiation; however,halometasone significantly aggravated the erythema reaction (P ＜ 0.05).Neither NSAIDs nor corticosteroids applied immediately after solar-simulated irradiation showed statistical effect on the degree of UV-induced erythema.When applied immediately after irradiation using the phototherapy device,butyl flufenamate 2.5％ ointment,butyl flufenamate 5％ ointment and halometasone ointment all induced a significant reduction in erythema reaction at 4 hours after 1 MED of irradiation (all P ＜ 0.05),and diclofenac caused a statistical decrease in erythema reaction at all the time points after 1-3 MED of irradiation (all P ＜0.05).Conclusions Topical use of butyl flufenamate before UV irradiation can effectively inhibit erythema reaction induced by 1-3 MED of irradiation.When applied immediately after irradiation,diclofenac shows the strongest erythema-suppressive effect,followed sequentially by butyl flufenamate and halometasone.

Objective To investigate the effect of fusidic acid cream on inflammatory reaction caused by skin barrier damage.Methods Eight male SKH-1 hairless mice were included in this study.The back of each of these mice were equally divided into six regions measuring 1 cm × 2 cm in size,which were then assigned into six groups:blank control group remaining untreated,barrier-impaired group,barrier-impaired and fusidic acid-treated group,barrier-impaired and vehicle-treated group,barrier-unimpaired and fusidic acid-treated group,barrierunimpaired and vehicle-treated group.Stratum corneum was removed by adhesive tape stripping to establish an animal model of acute skin barrier damage in the corresponding skin regions of these mice,and fusidic acid cream or vehicle was topically applied to the corresponding regions once.Twelve hours later,skin surface swab samples were collected from the back of these mice followed by bacterial culture and colony counting.Mice were then sacrificed,and skin tissue specimens were resected from these mice,and subjected to real-time fluorescence-based quantitative PCR for the measurement of the mRNA expressions of myeloid differentiation factor 88 (MyD88),interleukin-1α (IL-1α),IL-6,epidermal antibacterial peptides S100a8 and S100a9.Statistical analysis was carried out by repeated-measures analysis of variance (ANOVA) and least significant difference (LSD) test.Results The mRNA expressions of MyD88,IL-1α,IL-6,S100a8 and S100a9 were all significantly higher in the barrier-impaired group than in the blank control group (all P ＜ 0.05).Specifically,the mRNA expression level of MyD88 in the barrier-impaired group was 8 times that in the blank control group (8.3 ± 3.0 vs.0.8 ± 0.4).Compared with the barrier-impaired group,the barrier-impaired and fusidic acid-treated group showed a significant decrease in the mRNA expressions of IL-1α (2.8 ± 0.3 vs.20.1 ± 10.0,F =47.11,P ＜ 0.01),IL-6 (1.6 ± 2.3 vs.9.4 ± 4.0,F =16.18,P＜ 0.01),S100a8 (1.5 ± 1.4 vs.5.0 ± 1.6,F=59.71,P＜ 0.05) and S100a9 (1.2 ± 0.7 vs.3.4 ± 1.6,F=21.94,P ＜ 0.05).Conlusions Fusidic acid cream could attenuate the inflammatory reaction caused by acute skin barrier damage,which might partly explain its action mechanism in the treatment of inflammatory skin diseases.

Biomarkers, Tumor ; Humans ; Kidney ; Kidney Neoplasms ; Neoplasms

Objective To explore a new pathogenic gene mutationin in COL4A3 and COL4A4 genes of a family with thin-basement-membrane nephropathy (TBMN), and explain its mechanism.Methods Genomic DNA was extracted from blood samples.Mutation screening for all the exons in COL4A3 and COL4A4 of the proband was carried out by direct PCR sequencing.The sequences of the proband were compared with standard sequences in GenBank.After identifying the mutation in COL4A4, screening for the mutation site in 200 healthy controls and the rest of family members were conducted.RNA sequence of the proband was analyzed by reverse transcription PCR and TA cloning.The positive clones were sequenced for RNA screening.Results There was a G to A mutation in the 1459 site of COL4A4 (c.1459+G ＞ A) in the proband, her mother, and the elder sister, whereas the mutation was not found in other family members and healthy people.RNA screening showed that the COL4A4 (c.1459+G ＞ A) mutation was a heterozygous substitution in position + 1 of exon 21, in the splicing region.This mutation leaded to eliminating of exon 21 from the COL4A4 mRNA, causing the exon 21 deletion and frameshift mutation following the exon 20 in its amino acids sequence.Conclusions It is described that COL4A4 (c.1459+G ＞ A) is a new pathogenic mutation in TBMN, which further help understanding the pathogenesis and clinical diagnosis of TBMN.

Objective To explore the expression and the role of Toll-like receptor(TLR3 and TLR4) in rats with nephrotic syndrome induced by respiratory syncytial virus(RSV).Methods SD rats were inoculated intranasally and intraperitoneally with 6?106 plaque for-ming unit(PFU) RSV to construct RSV-induced nephropathy in rat model.Rats were anesthetized and blood was withdrawn from cardiac on day 4,14,30,60 after inoculation.The normal ones without intervention were set as control group.The renal histology was observed by light microscope and electron microscope.The urinary protein collected in 24 hours were measured.Meanwhile,the expressions of TLR3 and TLR4 were detected by indirect immunofluorescence staining and flow cytometry in peripheral blood mononuclear cells of rats.The results were analyzed by SPSS 13.0 softwore.Results After inoculation,the proteinuria increased and under the electron microscope the foot processes of glomerular epithelial cells were fused which resembled human minimal change nephrotic syndrome.Proteinuria reached the peak and the fusion of foot processes were most extensive in rats of RSV at 60 d.The expressions of TLR3 and TLR4 in each group of RSV-induced nephropathy in rat models were significantly higher than those in normal control group(Pa0.05).Conclusions TLR3 and TLR4 in peripheral blood mononuclear cells of RSV-induced nephropathy rat mo-dels had being significantly activated until 60 d after RSV inoculation.TLR signaling pathway may play an important role in nephrotic syndrome of rats induced by RSV.

0.05).3.Under light microscope, there was no change of the glomeruli detected in all of the LMWH-treated groups compared with the controls,while congestion and swel-ling in part glomeruli of group RSV were observed significantly. 4.Under electron microscope,glomerular structures of the LMWH-treated groups were almost normal compared with the control, while extensive foot process effacement was observed in group RSV under an electron microscope. 5.RSV RNA signal expressed weaker in the LMWH-treated groups than in group RSV.Conclusions Positive charged RSV destroys the glome-rular filtration barrier through its electrostatic interaction with the negative charged heparan sulfate(HS) of GBM. LMWH, as the analog of HS, charged with anion, competes with GBM HS to combine with RSV to keep the glomeruli from being infected and destroyed, and then reduce the proteinuria.

AIM:To explore the diagnosis and treatment methods of radioaction-induced optic neuritis ( RION) through the clinical dates of 17 patients.
METHODS: It was a retrospective case series study. From August 2008 to October 2013, 17 cases (24 eyes) of Rion clinical dates from Chinese PLA General Hospital were studied. The diagnosis methods including visual acuity, pupil, fundus, visual field, fundus fluorescein angiography (FFA), visual electrophysiological testing, and head MRI. To analysis the clinical date of patients with diagnosis of RION by statistical description.
RESULTS: The deterioration degree of vision: 13 eyes were classified as Ⅳ, 9 eyes as Ⅲ, 2 eyes as II. Ten eyes RAPD ( + ) , visual electrophysiology is extinguished. The retina of 5 eyes showed flame hemorrhages and cotton wool spots exudation. Optic nerve head edema in one eye. T1 - weighted MRI enhanced in 19 eyes which showed optic nerve of the intracranial and intratubal segments abnormal changed, optic chiasm and pituitary stalk signal abnormalities and enhancement of the optic nerve. Tortuous optic nerves and rough edges were observed in 5 eyes. Treatment effect: 4 eyes of visual acuity improved, 1 eye from blindness to light perception,1 eye from 0. 08 to 0. 2, 1 eye from 0. 4 to 0. 6,1 eye from 0. 04 to 0. 15, the rest of the cases did not see any improvement.
CONCLUSION: The unique clinical manifestation of RION can provide objective basis for clinical diagnosis in time, but there have not been proven any effective treatments.

Adult ; Aged ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Male ; Middle Aged ; Thymidine ; analogs & derivatives ; therapeutic use ; Treatment Outcome ; Young Adult

OBJECTIVETo investigate the correlation between human neutrophil peptide (HNP) and spontaneous bacterial peritonitis (SBP) in order to assess the diagnostic value of quantitative measurement of these alpha-defensins.

METHODSSeventy-seven patients with cirrhosis and ascites were divided into two groups according to diagnosis of SBP (n = 45 with SBP and n = 32 without SBP). Twenty-eight healthy individuals were analyzed as controls. HNP was detected by double-antibody sandwich assay. Peripheral white blood cell (WBC) counts, neutrophil ratio, and levels of procalcitonin (PCT) and C-reactive protein (CRP) were determined by standard methods. Receiver operating characteristic (ROC) curves were used to compare the diagnostic values of HNP, PCT and CRP in SBP.

RESULTSThere were no significant differences between the three groups (SBP, non-SBP, and healthy controls) for WBC count ((6.01+/-2.25)*109 /L, (4.85+/-1.94)*109 /L, and (5.49+/-1.76)*109 /L; F = 2.91, P more than 0.05) and neutrophil ratio (70.70%+/-10.42%, (68.20%+/-8.97%, and 69.50%+/-8.69%; F = 3.07, P more than 0.05). However, compared to the non-SBP group and the healthy controls, the SBP group showed significantly higher levels of HNP ((9.99+/-3.33) ng/ml and (8.92+/-2.30) ng/ml vs. (17.66+/-6.40) ng/ml; q = 3.20 vs. 3.52, P less than 0.05), serum CRP ((15.08+/-9.95) ng/ml and (5.96+/-2.91) ng/ml vs. (31.32+/-18.65) mg/L; q = 11.03 vs. 3.69, P less than 0.05), and positive rate of PCT (25.0% and 10.0% vs. 62.2%; X2 = 10.41 vs. 15.40, P less than 0.0125). The areas under the curve (AUC) showed the following descending trend: HNP more than PCT more than CRP (0.719, 0.707, and 0.629 respectively). Using cut-off points of 10 ng/ml for HNP, 0.5 ng/ml for PCT, and 8 mg/L for CRP, the respective sensitivities for diagnosis of SBP were 71.1%, 62.2%, and 73.3%, the respective specificities were 71.9%, 75.0%, and 56.3%, and the respective Youden's indexes were 0.430, 0.372, and 0.296.

CONCLUSIONHNP is closely related to SBP and can diagnose SBP as reliably as PCT. CRP may help to diagnose SBP, but the results from routine blood testing did not show sufficient statistical significance for diagnosing SBP.

Adult ; Aged ; Bacterial Infections ; blood ; diagnosis ; C-Reactive Protein ; metabolism ; Calcitonin ; blood ; Calcitonin Gene-Related Peptide ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Peritonitis ; blood ; diagnosis ; microbiology ; Protein Precursors ; blood ; alpha-Defensins ; blood