Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

Abstract?AlM:To investigate the influence of lOL refractive index ( Rl ) on measurement of straylight following cataract surgery.?METHODS:ln this case-control study, 77 eyes of 77 age-related cataract patients who underwent cataract phacoemulsification with posterior chamber aspherical arylic lOL implantation surgery in the Eye Hospital of China Medical University from Aug 2013 to Mar 2014, with a best corrected visual acuity ( BCVA) of 0. 5 or better, were classified into 3 groups randomly using 3 types of lOL: Tecnis ZCB00 Group ( Rl = 1. 47, 22 eyes of 22 subjects); Hoya PY60AD Group ( Rl=1. 52, 24 eyes of 24 subjects);Alcon SN60WF or lQ Group ( Rl=1. 55, 31 eyes of 31 subjects ) . BCVA, pupil size, astigmatism, axial length, intraocular straylight were measured respectively.? RESULTS: Age, axial length, BCVA, pupil size, astigmatism of the three groups were not significant difference (P>0. 05). The straylight of Tecnis, Hoya, lQ group were 1.04±0. 15, 1. 19±0. 14, 1. 14±0. 18. Straylight levels had significant differences among three groups ( F=5. 352, P = 0. 007 < 0. 05 ). There was no significant correlation between BCVA and straylight value (r=-0. 133,P=0. 124>0. 05).?CONCLUSlON:Patients chosen the higher Rl lOL may have a higher straylight level after the surgery.

Objective： To observe the effect of Qingkailing (QKL) on brain damage induced by glutamate, in order to seek for effective drugs for antagonizing neurotoxicity of glutamate. Methods：The number and morphological metrology of neurocytes in cerebral cortex and hippocampus were detected by MIAS-300 image analyser, electron microscope and immunohistochemical methods. Results：QKL could alleviate the glutamate induced accumulation of water and sodium in brain tissue，relieve the metrological and structural damage of cerebral cells in cortex and hippocampus, reduce the percentage of c-fos positive cell in brain. Conclusion： QKL could protect brain damage induced by glutamate, which might be related to the inhibition of QKL on the enhancement of c-fos gene expression induced by glutamate.

OBJECTIVETo study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.

METHODST-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well. All clinical data of patients after HSCT were collected and studied. Survival rates of patients after HSCT were estimated with Log-rank test. Univariate and multivariate analysis of prognostic factors were carried out by COX's proportional hazard regression model.

RESULTSThe mean value of TREC in normal donors was (3351 +/- 3711) copies/10(5) cells. There was an inverse correlation between TREC and age in the donor groups. Before transplantation, all patients were detected TREC, with a mean TREC number of (180 +/-332) copies/10(5) cells being significantly lower than that of normal donors. The results of univariate analysis showed that the counts of pre-HSCT TREC were closely, correlated with long term survival and chronic graft versus host disease (cGVHD) (P < 0.05) and with CMV infection (P = 0.084) but not with acute graft versus host disease (aGVHD). The results of multivariate analysis showed the same thing as that of univariate analysis.

CONCLUSIONPretransplantation host thymic recent output function is closely correlated with prognosis in MSD-BMT and can be a factor for predicting the outcome of HSCT.

Adolescent ; Adult ; Child ; Female ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Male ; Middle Aged ; Prognosis ; Receptors, Antigen, T-Cell ; genetics ; Retrospective Studies ; Siblings ; Thymus Gland ; immunology ; Transplantation, Homologous ; immunology ; methods

OBJECTIVETo explore the effect of CMV gB genotypes on viral load and treatment time in patients with CMV infection after hematopoietic stem cell transplantation (HSCT).

METHODSViral load was detected by real-time (RT) quantitative polymerase chain reaction (PCR) (Q-PCR), CMV gB genotypes by PCR restriction fragment length polymorphism (RFLP) (PCR-RFLP) in 115 patients with CMV infection (CMV-DNA positive) after HSCT during July 2004 and May 2010.

RESULTS(1) The distribution of CMV gB genotypes in HSCT recipients were as following: gB1, 42/115 (36.52%); gB2, 3/115 (2.61%); gB3, 43/115 (37.39%); gB4, 2/115 (1.74%). 20 patients (17.39%) had a combination of 2 different CMV genotypes and 5 patients (4.35%) had a CMV variant that lacked an RsaI digestion site, herein named gB5. (2) The median viral load were 2.7×10(3)(1.81×10(3) ∼ 6.03×10(4)) in gB1, 4.0×10(3) (1.32×10(3) ∼ 6.39×10(4)) in gB3 and 1.2×10(4)(2.28×10(3) ∼ 6.50×10(5)) in mixed gB. There was no statistical difference in viral load between gB1 and gB3 (P > 0.050). There was significantly statistical difference in viral load between single-gB (gB1 or gB3) and mixed-gB (P < 0.05). (3) The median treatment time was 17 days in mixed-gB and 14 days in single-gB. There was significantly statistical difference between two groups (P < 0.05). Conclusion gB genotype may have an impact on CMV DNA load and treatment time in HSCT recipients with CMV infection.

Adolescent ; Adult ; Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; virology ; DNA, Viral ; isolation & purification ; Female ; Genotype ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Viral Envelope Proteins ; genetics ; Viral Load ; Young Adult

Objective:To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs).Methods:The HMCs were cultured.The subcellular location of IgA was detected by immunofluorescence staining;the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing.The expressions of Igαt and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction.The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose.The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) data-base.Results:By immunofluorescence staining,we detected the presence of IgA heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λ mRNA in the NCBI database exhibited that the similarities were 99％,97％,98％ and 97％,respectively.Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant.To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry.The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No.52 and No.104,amino acids between No.154 and No.221,amino acids between No.276 and No.327 from Ig Cα1 and amino acids between No.52 and No.113,amino acids between No.151 and No.204,amino acids between No.251 and No.314 from Ig Cα2 were the same with those derived from B cells.Conclusion:Our findings suggested that HMCs could synthesize and secret IgA.

OBJECTIVE Discussion on the application of GC-MS to study the metabolic fingerprinting of serum in pneumo-nia group with syndrome of wind-cold in order to provide data support for the quantitative study of syndromes in TCM.METH-ODS In the study of patients with wind-cold(WC)in 30 cases,the normal healthy(NH)in 30 cases,Serum samples were col-lected and serum metabolites profiles were analyzed by GC-MS coupled with a couple of statistical analyses.RESULTS Clear separations between WC and NH existed.Differential metabolites and several metabolic pathways related organic acids,amino acids,fat were identified.Compared with NH group,the levels of organic acids,amino acids,fat were upregulated,such as L-Alanine,L-Aspartic acid,L-Glutamic acid,Glycine,Serine,L-Threonine,Taurine,L-Lysine,and D-2-Aminobutyric acid, Glycolic acid,Glycerol,1-Monopalmitin,cholesterol,d-Glucose were down in serum with WC group.And,five abnormal met-abolic pathways,such as Alanine metabolism,Glycine metabolism,Taurine metabolism,aa-tRNA biosynthesis and Lysine deg-radation were identified.CONCLUSION This study detected abnormal metabolites and pathways in WC,which may be due to multiple energy metabolism pathways-virus infected;taking advantage of metablomic techniques,we may establish Character-ized Spectrum Database in pediatric viral pneumonia and syndrome differentiation pattern standardization of TCM so as to pro-vide the useful methods in study of syndrome standardization.

Objective To elucidate the genetic diversifications of avian influenza subtype H5N1 viruses in the boundary regions of Yunnan province during 2009 to July,2011.Methods Swab samples were collected from foreign poultry and wild birds in boundary regions of Yunnan province during 2009 to July,2011 and tested by H5/N1 subtype-specific multiplex RT-PCR.The HA genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD 18-T vectors for sequencing.Both alignment and phylogenetic analysis were performed with sequences of the known reference strains.Results Fifteen different HA sequences were obtained from 36 representative positive samples and could be divided into 2 distinct Clades (2.3.2 and 2.3.4).Through phylogenetic analysis,Clade 2.3.2 and 2.3.4 could then be further divided into 3 ( Ⅱ -1 to Ⅱ -3 ) and 2 smaller clades ( Ⅰ -1 and Ⅰ -2),respectively.The viruses of Clade 2.3.2 Ⅱ -1 and Ⅱ-2 were new variant strains of H5N 1 virus.The cleavage sites of HA from positive samples all possessed molecular characterization of highly pathogenic avian influenza virus.Mutation of key amino acids had been found among receptor binding sites,potential glycosylation sites,neutralizing epitopes and others.Conclusion It seemed evident that the H5N1 subtype viruses showed genetic diversifications and had undergone the evolution progress of multi-clade (2.3.2,2.3.4) to single caide (2.3.2) in the boundary regions of Yunnan province,during 2009 to July,2011.

Objective: To investigate the safety and efficacy of pulmonary vein deployment technique for percutaneous closure of atrial septal defects (ASD) solely under echocardiography guidance. Methods: A total of 38 ASD patients received pulmonary vein deployment in our hospital from 2012-10 to 2016-09 since the conventional method could not deliver the occluder to correct place. The patients were with the mean age at (16.0±15.6) years, body weight at (37.2±22.9) kg and ASD diameter at (17.1±4.2) mm. Operative effect was assessed by echocardiography. Follow-up study was conducted at 1, 3, 6, 12 months post-operation and at each year thereafter. Results: 37 patients were successfully finished pulmonary vein deployment for percutaneous closure of ASD solely under echocardiography guidance. One patient was successfully treated by a controlled steerable sheath. The mean operative time was (25.2±5.1) min and mean diameter of ASD occluder was (22.9±5.6) mm. 2 patients had trivial residual shunt at the early post-operative stage. No peripheral vascular injury, pulmonary vein and cardiac perforation occurred. All 38 patients were recovered and discharged. The average in-hospital time was (2.9±0.7) days. The patients were followed-up for (23.9±15.4) months, without complications of residual shunt, pericardial effusion, aortic regurgitation and pulmonary vein stenosis. Conclusion: Pulmonary vein deployment technique for percutaneous closure of ASD solely under echocardiography guidance was safe and effective; it can avoid radiation damage and provided a simple and practical method for ASD patients who failed to conventional method under echocardiography guidance.

OBJECTIVETo explore the possible role of CD28/CTLA-4 co-stimulators in immune pathophysiology of acquired aplastic anemia(AA).

METHODSBy FACS, the percentages of CD28, CTLA-4 expressing CD3+ CD4+ T cells and the level of Th1, Th2 in bone marrow were detected in 23 AA patients at active phase, 10 at recovery phase and 15 normal controls. The relationship between the co-stimulators, Th1, Th2, and absolute neutrophil counts (ANC) was evaluated.

RESULTS(1) The percentage of CD28 and CTLA-4 expressing CD3+ CD4+ T cells in bone marrow, and CD28+/CTLA-4+ ratios were (31.40 +/- 10.83)%, (2.45 +/- 1.30)% , and 17.02 +/- 13.44 in normal controls respectively, (39.84 +/- 10.89)%, (1.43 +/- 0.67)%, and 43.04 +/- 37.61 in AA at active phase, respectively, (22 +/- 9.08)%, (3.46 +/- 2.26)%, and 10.49 +/- 7.8 in AA at recovery phase, respectively. The percentage of CD28 and CD28+/CTLA-4+ ratio were significantly higher, while CTLA-4 were lower in active phase AA patients than in normal controls (P < 0.05). These values in recovery phase AA were comparable to those in normal controls. (The Th1, Th2, and Th1/Th2 in bone marrow were (4.21 +/- 2.11)%, (1.99 +/- 1.27)%, and 2.46 +/- 1.28 in normal controls respectively, (11.13 +/- 4. 96)%, (2.46 +/- 1.65)%, and 5.20 +/- 1.98 in active phase AA and (5.39 +/- 4.2)9%, (2.53 +/- 2.41)%, and 2.87 +/- 1.43 in recovery phase AA, respectively. The percentage of Th1 and Th1/Th2 ratio were significantly higher in AA patients at active phase than in normal controls (P < 0.05). (3) The CD28+/CTLA-4+ ratio was positively related to the Th1+ /Th2+ ratio (P < 0.05). ANC was negatively related to CD3+ CD4+ CD28+ T cells (P < 0.01), and positively to CD3 + CD4 ' CTLA-4' T cells (P < 0.01) respectively.

CONCLUSION(1) The expression of CD28 was increased while CTLA-4 decreased on the membranes of CD3+ CD4+ T cells in bone marrow of AA patients. (2) The abnormal expression of CD28 costimulator promoted the shift of immune balance to Thl type. (3) The unbalance of CD28+ / CTLA-4+ is important for the immune pathophysiology of AA.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; immunology ; metabolism ; Antigens, CD ; immunology ; metabolism ; CD28 Antigens ; immunology ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; CTLA-4 Antigen ; Child ; Female ; Humans ; Male ; Middle Aged ; Th1 Cells ; immunology ; Th2 Cells ; immunology