Objective To establish a rapid assay with high sensitivity and specificity based on the sequences for group specific gene (GS) and pathogenicity island pag A gene.Methods The PCR primers and probes were designed after the whole sequence was systemically analyzed with bio-informafion tools and blasted with Genebank database.The amplicons were inserted into plasmids so that they could be used as the standard templates to evaluate the sensitivity of the diagnostic system.This assay was based on TaqMan probes and portable Smartcycle PCR machine.Results The detection level was approximately 100 copies per reaction.There was no cross-reaction with other species of Bacillus.This assay could be completed in one hour in laboratory.Conclusion The duplex TaqMan PCR assay could be used to detect Bacillus anthracis rapidly with high sensitivity and specificity.

Adult ; Aged ; Decompression, Surgical ; Female ; Humans ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged ; Nerve Block ; methods ; Spinal Nerve Roots ; Spinal Stenosis ; surgery

OBJECTIVETo analyze the relationship between high-performance liquid chromatography (HPLC) fingerprints of the chloroform extract fractions of Peucedanum harrysmithii var. subglabrum (PHS) and its phlegm-reducing effect, in order to establish "active component group for reducing phlegm".

METHODHPLC was adopted to determine and analyze HPLC fingerprints of chloroform extract fractions of PHS. Phenol red expectorant experiment was used to observe the phlegm-reducing effect in mice. Mice were administered intragastrically with chloroform extract fractions for 6 days (1.4 g x kg(-1)), with acute bronchitis syrup as the positive control drug (12 mL x kg(-1)). The phenol red secretion in mice was determined by spectrophotometer. Then the grey relational analysis was used to study the spectrum-effect relationship.

RESULTThe phlegm-reducing effect of the chloroform extract fractions of PHS were resulted from the combined effect of all of its chemical components. Its various characteristic peaks represented different chemical components, and the order of their contributions to the phlegm-reducing effect was (number of peaks) 13 > 12 > 16 > 18 > 19 > 6 > 20 > 14 > 1 > 11 > 15 > 10 > 17 > 2 > 5 > 4 > 7 > 3 > 8 > 9, in No. 1, 3, 4, 10, 13 and 16 characteristic peaks were identified as marmesin, psoralen, xanthotoxin, Pd-Ib, pteryxin and peuformosin.

CONCLUSIONThe chloroform extract fractions of PHS show strongly phlegm-reducing effect. There may be certain relationship between their HPLC fingerprint and phlegm-reducing effect.

Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Ferns ; chemistry ; Mucus ; drug effects

OBJECTIVETo establish a HPLC method for determination two constituents in bark of Paeonia Suffuticosa.

METHODThe reversed phase HPLC system consisting of an Alltima ODS column (4.6 mm x 150 mm, 5 microm) and a mixture of water-THF-methanol-HAc (60:20:20:0.05) as the mobile phase was used. The flow rate was 0.8 mL x min(-1) and UV detection was set at 274 nm.

RESULTThe assay displayed good linearity over the concentration ranges of 0.06-1.0 microg (r = 0.999 9, gallic acid) and 0.16-2.58 microg (r = 0.999 9, paeonol) respectively. The average recoveries (n = 9) of gallic acid and paeonol were 98.6% (RSD = 3.0%), 98.2% (RSD = 2.5%), respectively. The samples were extracted with methanol for 24 h bu maceration.

CONCLUSIONThe method is simple, accurate and can be used for the quality study of bark of P. suffruticosa.

Acetophenones ; analysis ; Chromatography, High Pressure Liquid ; methods ; Gallic Acid ; analysis ; Paeonia ; chemistry ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control

BACKGROUNDTrichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.

METHODSHaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.

RESULTSHaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.

CONCLUSIONThe cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.

Cells, Cultured ; Cytokines ; biosynthesis ; Humans ; Keratinocytes ; immunology ; Lectins, C-Type ; genetics ; physiology ; RNA, Messenger ; analysis ; Receptors, Pattern Recognition ; genetics ; physiology ; Toll-Like Receptor 2 ; physiology ; Trichophyton ; immunology

BACKGROUNDCathepsin B plays an important role in cell cycle, extracellular matrix changes and cutaneous tumorigenesis: whether it plays a role in photoaged skin remains unknown. This study aimed to investigate the role of cathepsin B in skin photoaging in vivo and in vitro.

METHODSThe expressions of cathepsin B were compared with immunohistochemical methods in solar exposed skin and solar protected skin of six healthy Chinese volunteers. The mRNA and protein expression of cathepsin B in ultraviolet light A (UVA) induced premature senescence fibroblasts in vitro were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting technique.

RESULTSDecreased expression of cathepsin B was observed in photoaged skin compared with that of the solar protected skin. In the UVA induced, premature senescence fibroblasts, a lower expression of cathepsin B was detected by Western blotting and a decreased synthesis of cathepsin B mRNA in the same cells was revealed by real-time RT-PCR.

CONCLUSIONSThe results demonstrated a significant negative correlation between skin photoaging and cathepsin B in vitro and in vivo. We propose that cathepsin B, besides matrix metalloproteinases and antioxidant enzymes, is involved in the process of skin photoaging in that it contributes to extracellular matrix remodelling and is a dominant protease in cellular apoptosis and senescence.

Blotting, Western ; Cathepsin B ; analysis ; genetics ; physiology ; Female ; Fibroblasts ; radiation effects ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Skin ; radiation effects ; Skin Aging ; Ultraviolet Rays ; beta-Galactosidase ; analysis

Using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, macroporous adsorbent resin, and reversed-phase HPLC, 115 compounds including diterpenes, sesquiterpenes, treterpenes, coumarins, lignans, fatty acid derivatives, and simple aromatic derivatives were isolated from an ethanol extract of branch of Fraxinus sieboldiana (Oleaceaue), and their structures of the compounds were elucidated by spectroscopic methods including 1 D, 2D NMR and MS techniques. Among them, 41 compounds were new. In previous reports, we have been described the isolation, structure elucidation, and bioactivities of the 41 new compounds and 22 known orii including 8 coumarins, 4 phenolic and 12 phenylethanoidal glycosides. As a consequence, we herein reported the isolation and structure elucidation of the remaining 50 known compounds including 8- hydroxy-12-oxoabieta-9(11),13-dien-20-oic 8, 20-lactone(1), 6beta-hydroxyfcrruginol(2),(+)-pisiferic acid(3), (+)-pisiferal(4),(+)-7-dehydroabiet6none(5), 1-oxomiltirone(6), subdigitatone(7), linarionoside B(8), (9S)-linarionoside B(9), (3R,9R)-3-hydroxy-7,8-dihydro-beta-ionol 9-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside(10), ursolic acid(11), betulinic acid(12), euscaphic acid(13), (+)-syringaresinol(14), (+)-fraxiresinol(15), (+)-1-hydroxysyringaresinol(16), pinoresinol(17), medioresinol(18), 8-acetoxypinoresinol(19), epipinoresinol(20), (-)-olivil(21), (+)-cyclo-olivil(22), 3,3'-dimethoxy-4,4',9-trihydroxy-7,9'-epoxylignan-7'-one(23),(+)-1-hydroxypinoresinol 4'-O-beta-D-glucopyranoside (24), (+)-1-hydroxypinoresinol 4"-O-beta-D-glucopyranoside(25),(+)-syringaresinol O-beta-D-glucopyranoside (26), liriodendrin (27), ehletianol D(28), icariside E5(29) (-)-(7R, 8R)-threo-1-C-syringylglycerol(30),(-)-(7R, 8S)-erythro-guaiacylglycerol (31),(-)-(7R, 8R)-threo-guaiacylglycerol(32), 3-(4-beta-D-glucopyranosyloxy-3-methoxy)-phenyl-2E-propenol(33),2,3-dihydroxy-l-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone(34), 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (35), 3-hydroxy-l-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone(36), omega-hydroxypropioguaiacone(37), sinapyladehyde(38), trans-p-hydroxycinnamaldehyde(39), syringic acid(40), vanilic acid(41), vanillin(42), 4-hydroxy-benzaldehyde (43), (24R)-24-ethyl-5alpha-cholestane-3beta,5,6beta-triol(44), beta-sitosterol(45), daucosterol(46), 2,6-dimethoxy-I,4-benzoquinone(47), 2,6-dimethoxy-pyran-4-one(48), 1-(beta-D-ribofuranosyl)uracil(49), and mannitol(50). Compouds 1-7,12,18,28-37,44 and 48 were obtained from the genus Fraxinus for the first time.
Fraxinus ; chemistry ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Plant Extracts ; analysis

Hematoma, Epidural, Spinal ; etiology ; Humans ; Liver Cirrhosis, Alcoholic ; complications ; Male ; Middle Aged

AIMTo study the effect of antiparallel phosphorothioate triplex-forming oligonucleotide (apsTFO) matching with the shear stress response element (SSRE) of tissue factor (TF) gene promoter region on the expression of TF in endothelial cells (ECs) of rat common carotid artery stenosis.

METHODSThe model of common carotid artery middle segment stenosis was established by silica gel pipe loop ligation in SD rats. The mRNA expression and protein synthesis of TF, early growth response-1 (Egr-1) and specificity protein 1 (Sp1) were measured by in situ hybridization (ISH) and immunohistochemistry (IHC) technique. GT21-apsTFO, GT20-apsTFO, GT20-psTFO and FITC-labeled apsTFO, matching with the SSRE of TF gene promoter region, were designed, and intravenously injected into rats at 0.5 h before operation. TFO was detected 4 h after the operation, and the mRNA expression and protein synthesis of TF, Egr-1 and Sp1 were detected 6 h after the operation.

RESULTSThere were much fluorescence in vascular tissue, especially in the nuclear of ECs 4.5 h after the injection of apsTFO. The mRNA expression and protein synthesis of TF reduced by 22% - 23% with injection of GT20-apsTFO 6.5 h after stenosis (P < 0.01) and by 10% - 11% with GT21-apsTFO at the same time (P < 0.05). The inhibition by GT20-apsTFO was stronger than that of the GT21-apsTFO (P < 0.05). The expression of TF was not inhibited by the GT20-psTFO (P > 0.05). The mRNA expression and protein synthesis of Egr-1 and Sp1 did not change in the rat treated with GT20-apsTFO, GT20-psTFO and GT21-apsTFO (P > 0.05).

CONCLUSIONapsTFO could mero-inhibit the expression of TF gene but could not change the expression of Egr-1 and Sp1 protein.

Animals ; Carotid Stenosis ; genetics ; metabolism ; pathology ; Early Growth Response Protein 1 ; genetics ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression ; drug effects ; Immunohistochemistry ; In Situ Hybridization ; Male ; Oligonucleotides ; chemical synthesis ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Shear Strength ; Sp1 Transcription Factor ; genetics ; metabolism ; Stress, Mechanical ; Thromboplastin ; genetics ; metabolism

Objective: To compare the time of the recovery of neutrophils or leukocytes by pegylated recombinant human granulocyte stimulating factor (PEG-rhG-CSF) or common recombinant human granulocyte stimulating factor (rhG-CSF) in the myelosuppressive phase after induction chemotherapy in newly diagnosed acute myeloid leukemia (AML) patients. At the same time, the incidences of infection and hospitalization were compared. Methods: A prospective randomized controlled trial was conducted in patients with newly diagnosed AML who met the enrollment criteria from August 2014 to December 2017. The patients were randomly divided into two groups according to a 1:1 ratio: PEG-rhG-CSF group and rhG-CSF group. The time of neutrophil or leukocyte recovery, infection rate and hospitalization interval were compared between the two groups. Results: 60 patients with newly diagnosed AML were enrolled: 30 patients in the PEG-rhG-CSF group and 30 patients in the rhG-CSF group. There were no significant differences in age, chemotherapy regimen, pre-chemotherapy ANC, WBC, and induction efficacy between the two groups (P>0.05) . The median time (range) of ANC or WBC recovery in patients with PEG-rhG-CSF and rhG-CSF were 19 (14-35) d and 19 (15-26) d, respectively, with no statistical difference (P=0.566) . The incidences of infection in the PEG-rhG-CSF group and the rhG-CSF group were 90.0%and 93.3%, respectively, and there was no statistical difference (P=1.000) . The median days of hospitalization (range) was 20.5 (17-49) days and 21 (19-43) days, respectively, with no statistical difference (P=0.530) . Conclusions: In AML patients after induction therapy, there was no significant difference between the application of PEG-rhG-CSF and daily rhG-CSF in ANC or WBC recovery time, infection incidence and hospitalization time.
Granulocyte Colony-Stimulating Factor/therapeutic use* ; Humans ; Induction Chemotherapy/adverse effects* ; Leukemia, Myeloid, Acute/drug therapy* ; Neutropenia ; Neutrophils ; Prospective Studies ; Recombinant Proteins