Objective To establish a method of TRFIA with high sensitivity and broad detecting range for serum HBV large surface protein (HBV-LP).Methods The monoclonal antibody of HBV-LP was covered on the microwell plate and incubated with HBV-LP in blood sample,then Eu3+ labeled antibody of HBs was added.HBV-LP in standard substance,blood samples of 66 chronic hepatitis B patients and 30 healthy controls was detected by the TRFIA and ELISA.x2 test and linear correlation analysis were used for data analysis.Results The dose-response curve of standard substance with TRFIA had good linear correlation (r=0.999).Normal reference range was established at 0-1.36 mg/L based on the ELISA results of 30 healthy controls.The sensitivity was 0.10 mg/L.The specificity was 100％ (30/30).Correlation coefficient between the TRFIA and the ELISA was 0.800 9 (P＜0.001).The positive detecting rates of the 2 methods were significantly different (89.4％(59/66) vs 77.3％(51/66),x2 =6.13,P＜0.01).The recovery rate for HBV-LP was between 95.93％-107.62％.The effective detecting range(CV＜10％) of TRFIA was 1.35-2 764.00 mg/L,and that of ELISA was 10.8-691.0 mg/L.Conclusion The TRFIA was established for HBV-LP detection with higher sensitivity and wider detecting range compared to ELISA.It has potential value for HBV screening and monitoring of antiviral therapy.

We describe the prenatal diagnosis and postnatal follow-up of a fetus with a pericallosal lipoma diagnosed by MRI. The pregnant woman was admitted to Zhongnan Hospital of Wuhan University in February 2019. MRI examinations with multiple sequences and imaging methods were performed prenatally at 31 +6 and 38 +3 weeks of gestation, and at two days and three months postnatally. The MRI images showed that the corpus callosum was normal and the lesion was located around the corpus callosum, with high signal intensity on T1 weighted imaging, medium-to-high signal intensity on T2 weighted imaging, and low signal intensity on fat suppression sequence. No abnormal blood vessels were found on susceptibility-weighted imaging and time-of-flight magnetic resonance angiography sequences. Based on the prenatal and postnatal MRI findings, the tumor was clinically diagnosed as a pericallosal lipoma.

Objective To evaluate changes of regional cerebral metabolism by proton MR spectroscopy (~1H-MRS) in patients with minimal hepatic encephalopathy (MHE) and to correlate these changes with the neuropsychological test. Methods Fifty-four patients with cirrhosis including nine patients with hepatic encephalopathy (HE),23 patients with MHE,22 patients without HE and 13 controls underwent neuropsychological tests and ~1H-MRS scanning. The volumes of interest included occipital gray matter and left parietal white matter regions. Ratio of spectral peak areas of N-acetylaspartate (NAA),choline (Cho),myo-inositol (mI),and glutamine/glutamate (Glx) relative to creatine (Cr) were acquired. Statistical analysis was conducted using independent t test and one-way analysis of variance. The results of different groups were compared by using the nonparametric Mann-Whitney U test with Bonferroni correction. Correlations among the ~1H-MRS ratios, the grade of HE, neuropsychological test and ammonia data were calculated with Spearman correlation test. Results The ratios of NAA/Cr,Cho/Cr,mI/Cr,Glx/Cr of the occipital gray matter and left parietal white matter regions in patients with cirrhosis are 1.55±0.12,0.48±0.10,0.42±0.14,2.52±0.48 and 1.73±0.17,0.75±0.16,0.42±0.16,2.75±0.59respectively,and they are 1.53±0.10,0.48±0.09,0.51±0.11,2.20±0.39 and 1.69±0.15,0.82±0.14,0.53±0.12,2.40±0.40 in patients without HE,1.58±0.13,0.48±0.08,0.38±0.13,2.62±0.39 and 1.78±0.18,0.74±0.14,0.38±0.15,2.84±0.58 in patients with MHE,1.54±0.12,0.50±0.13,0.29±0.07,3.04±0.31 and 1.70±0.19,0.62±0.16,0.29±0.07,3.37±0.38 inpatients with HE.Compared with controls, decreased mI/Cr and Cho/Cr ratios and elevated Glx/Cr ratios were found in patients with cirrhosis (t=3.196,9.394,-6.527,P＜0.01,occipital gray matter. t=5.592,9.717,-6.681,P＜0.01,left parietal white matter＝ and in subgroup of patients without HE, with MHE and HE (F=5.097,25.896,20.204,P＜0.01,occipital gray matter.F=16.435,28.660,21.283,P＜0.01,left parietal white matter).Significant difference in these metabolic alterations was also found among the different groups of cirrhosis especially the ratios of Glx/Cr in occipital gray matter and left parietal white matter (P＜0.0084).The ratios of mI/Cr also significantly altered between patients without HE and with MHE (P＜0.0084).There was a significant negative correlation between the ratios of Cho/Cr,mI/Cr and the grade of HE (P＜0.01＝ and a significant positive correlation between the ratios of Glx/Cr and the grade of HE (r=0.709,P＜0.01,occipital gray matter; r=0.720,P＜0.01,left parietal white matter＝.NCT-A and DST of controls is (49±8) s and 39±6.They are (134±37),(83±26),(64±22) second and 15±2,25±9,35±8 in patients with HE,MHE and without HE respectively.The metabolic alterations of Cho/Cr,mI/Cr,Glx/Cr correlated significantly with neurepsychological tests in all subjects (P＜0.01＝.There was a significant positive and a negative correlation between the ratios of Glx/Cr and the data of NCT-A and DST respectively (r=0.570,-0.642,occipital gray matter; r=0.541,-0.632,left parietal white matter).The metabolic alterations of Glx/Cr had no correlation with ammonia data as well as other metabolic alterations.Conclusions ~1H-MRS study shows cerebral metabolic alterations of gray and white matter in patients with cirrhosis,especially the reduction in mI/Cr ratio and increase in Glx/Cr ratio. These changes correlate well with the neuropsychological tests and may be useful in predicting the presence of MHE.

Objective To explore the changes of S-100 protein(S-100) levels in blood in newborn rats with bilirubin encephalopathy.Methods The model was established by administered bilirubin intraperitoneally(200 mg/kg) in newborn rats.The blood and brains samples were taken from 8 rats at the sequential time points of 6,12,24,48,72 and 96 h after the model established.Eight normal rats were used as the control.The dynamic changes of S-100 were detected by enzyme linked immunosorbent assay(ELISA) and immunohistochemical technique.Results S-100 significantly increased in blood of bilirubin encephalopathy newborn rats compared with controls(P

Objective To investigate the inhibitory effect and the effect on cell proliferation and apoptosis of artemether(ART) on human gastric adenocarcinoma cell lines and pancreatic cancer cell lines in vitro. Methods The inhibitory effect of ART on human gastric cancer cell lines(SGC-7901 and MKN-45) and human pancreatic cancer cell lines(SW-1990 and BXPC-3) were detected by MTT assay,and the effect on cell cycle and apoptosis were evaluated by flow cytometry. Results It was indicated by MTT assay that the killing effect of ART on the cancer cell lines were positively correlated to time and dosage(P

Objective To study the mechanism of mast cell increase in cellular leiomyoma of uterus.Methods Tissue sections from 30 cases of cellular leiomyoma of uterus,15 cases of leiomyosarcoma and 30 cases of ordinary leiomyoma were studied using immunohistochemical double labeling techniques.The expression of mast cell tryptase ahd Ki-67 as well as mast cell tryptase and chemotactic factors RANTES,Eotaxin,monocyte chemoattractant protein-1(MCP-1),transforming growth factor-? (TGF-?)were double immunostained.Results Ki-67 in mast cells was rarely expressed in each group. Expressions of regulate upon activation normal T cell expressed and secreted(RANTES),Eotaxin and TGF-? in cellular leiomyoma were 78%,89%,91%,respectively.They were all higher than those in ordinary leiomyoma and leiomyosarcoma(P

Objective To compare the sub-cellular proteome of isoniazid ( INH)-resistant Mycobacterium tuberculosis (MTB) with that of sensitive strains for identifying of unique proteins of these strains and discussing their preliminary application in clinical diagnosis. MethodsProteins of cell wall and membrane of 5 INH-resistant strains and 5 INH-sensitive strains were extracted by density gradient centrifugation.The extracts were subsequently analyzed using weak cation exchange (WCX) liquid chromatography ( LC )followed reverse phase (RP) liquid chromatography to compare the sub-cellular protein patterns. A total of 1280 fractions were collected and identified by matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF MS/MS). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for cell component and biological process analysis. Normalized Spectral Abundance Factors (NASF) was used for semi-quantity of protein expression. 5 proteins significantly up-regulated in INH-resistant strains and 2 proteins significantly up-regulated in INH-sensitive strains were selected for ELISA analysis with autologous sera respectively. Results A total of 347 proteins were identified. Cell component analysis showed that 58％ proteins were cells well or membrane proteins. Biological process analysis showed that 31％ proteins involved in carboxylic/monocarboxylic acid biosynthetic and metabolic process, 26％ and 15％ proteins involved in organic acid or fatty acid biosynthetic and metabolic process,while 28％ proteins involved in lipid biosynthetic , metabolic, transport and localization process. O-succinylbenzoate synthase, monooxygenase, hypothetical protein Rv2255c, nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase and membrane phosphatidate cytidylyltransferase cdsA were up-regulated in INH-resistant strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-sensitive sera. The differences between the INH-resistant sera and the INH-sensitive sera were significant ( t = 0.028, 0.044, 0.066, 0.064, 0.083, all P＜0.01 ). Chain A of Rv2002 Gene Product and Chain A of Crystal Structure Of Rv2632c were up-regulated in INH-sensitive strains and fractions contained these proteins could elicit specific antibody response with autologous sera. The A450 was higher than that with INH-resistant sera. The differences between the INH-sensitive sera and the INH-resistant sera were significant (t=0.053, 0.073, both P＜0.05). ConclusionThe combination of density gradient centrifugation and 2D-LC MS/MS technology is useful in enrichment and identification of differential expressed proteins between INH-resistant and INH-sensitive strains at sub-cellular level. It is useful in finding antigens associated with INH-resistant MTB infection, which may prove useful for further study in the mechanism of INH resistant, as well as interaction between MTB and host.

Objective To study the activation of Janus protein tyrosine kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway and its inhibitor-signal transducer and activator of transcription-1(SOCS-1) in patients with systemic lupus erythematosus. Methods A total of 45 patients with active systemic lupus erythematosus (SLE) and 30 healthy controls were randomly selected. Western blot was performed to measure the expression of Stat1 protein and phospho-Stat1 protein (an activated form of Stat1 protein) in the monocytes after stimulation with recombinant high mobility group box1 (rHMGB1) at various time points. Expression of Stat1 protein in the skin or lesional skin was also detected. Phasic expressions of SOCS-1 mRNA in the monocytes after rHMGB1 stimulation were detected by real-time reverse transcription-polymerase chain reaction. SOCS-1 gene expression in the skin or lesional skin was also detected. Results The expression level of Stat1 proteins in the monocytes from patients with SLE was higher than that from healthy controls (t=9.16,P＜0.01) and positively correlated with SLE disease activity index (SLEDAI) (r=0.59,P＜0.01). Expression of phospho-Stat1 in the monocytes from SLE patients was time-dependently upregulated after stimulation with rHMGB1 at various time points, while expression of SOCS-1 mRNA remained unchanged(all P＞0.05). Expressions of phospho-Stat1 protein and SOCS-1 mRNA in the monocytes from healthy controls were increased transiently after stimulation with rHMGB1(all P＜0.05). Both expressions of phospho-Stat1 protein and SOCS-1 gene in the lesional skin from patients with SLE were upregulated compared with those in normal skin from healthy controls (all P＜0.01). Conclusion There are hyperactivation of JAK-STAT1 signaling pathway and negative feedback down-regulation of SOCS-1 in patients with systemic lupus erythematosus. HMGB-1 may be partly involved in the pathogenesis of SLE by the abnormal mediating function of JAK-STAT1 signal transduction pathway.

BackgroundThe quest to look for seed cells is a hot spot of cornea transplant research in solving the problem of the lack of donor. Bone marrow mesenchymal stem cells(BMSCs) have been successfully induced into retinal ganglion cells(RGCs) in vivo,but the successful induction of BMSCs into corneal endothelial cells has not been reported.Objective This experiment was to study the transplantation of BMSCs on corneal endothelial surface using the splitting Descemet's membrane. MethodsFour healthy adult rhesus monkeys were divided into the experimental group ( 3 monkeys) and control group ( 1 monkey). Mesenchymal stem cells (MSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. The cultured cells were identified by flow cytometry and its ability to differentiate was determined by allowing them to differentiate into adipocytes in vitro and labeled by 5-bromodeoxyuridine ( BrdU ) for subsequent identification. Corneal grafts of 7 mm in size with tearing of the Descemet' s membrane were prepared in the experimental group and control group. After labeling by 5-bromodeoxyuridine( BrdU ) ,cultured cells were transplanted onto the endothelial surface of cornea grafts in the experimental group, but no cultured cells were seeded in the graft of the control group. The corneal grafts were then sutured in situ, and were removed 1,2 or 3 months after operation to examine the distribution and connection between transplanted cells and their morphologic changes under the electron microscope. Results High purity MSCs were harvested by density gradient centrifugation combined with adhering method. Cultured cells reached confluency after 12 to 16 days, presenting with a spindle shape and parallel or swirling arrangement. Flow cytometry analysis showed that 94.26％ of cells were positive for CD29,7. 51％ for CD34 and 4. 02％ for CD45. Larger nuclei filled with plastosomes, golgiosomes and rough endoplasmic reticula were found on the graft under the transmission electron microscope( TEM ). After 3 weeks, MSCs were differentiated into adipocytes where Oil Red O staining resulted in an orange-red staining in the cytoplasm and blue staining in the nuclei. The transplanted cells attached loosely on the endothelial surface of the corneal graft and came in contact with each other in one month. The shape of the cells appeared as spindle-shaped and polygonal after 2 months and became tightly packed after 3 months. The positive cells retained the BrdU label and presented with brown nuclei. No endothelia cells grew in the cornea graft in the control group, with an absence of BrdU labeling. Conclusions Mesenchymal stem cells can be transplanted onto the corneal endothelial surface successfully and form a monolayer using the centrifugation method, and present with good survival and proliferation ability.

OBJECTIVETo clone the pokeweed anti-viral protein (PAP) gene, to express it in Pichia pastoris, and to study the inhibitory effect of PAP on U251 in vitro.

METHODSThe cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca americana. The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastoris GSI15 after methanol induction. SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant. The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis.

RESULTSThe full-length PAP gene was cloned. The recombinant expression plasmid pPICZaA-PAP was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass (M) of the recombinant protein was about 35 kDa. The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis. PAP possessed very high ability to inhibit the growth of U251. The anti-tumor activities (IC50) to U251 cells of PAP was 81.0 microg/mL.

CONCLUSIONPAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.

Antineoplastic Agents, Phytogenic ; biosynthesis ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Humans ; Pichia ; metabolism ; Ribosome Inactivating Proteins, Type 1 ; biosynthesis ; pharmacology