Significance of Mannan Binding Lectin-Associated Serine Protease 2 in Urinary Extracellular Vesicles in IgA Nephropathy.

PURPOSE: Immunoglobulin A (IgA) nephropathy (IgAN) is a common chronic glomerulonephritis and the main cause of end-stage renal diseases. Recent evidence suggests that mannan binding lectin associated serine proteases 2 (MASP2) is related to IgAN; therefore, we investigated the expression and significance of MASP2 in serum and urinary extracellular vesicles (UEVs) in patients with IgAN. METHODS: Thirty-eight patients with IgAN and 17 healthy controls were enrolled in this study. UEVs were extracted by ultracentrifugation. The separation by ultra-high-speed centrifuge was verified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Candidate internal references (TSG101, CD9, flotillin, ß-actin and GAPDH) were identified by western blotting in the control group, and the expression of MASP2 in the UEVs was compared. The levels of MASP2 in the serum and UEVs in the IgAN and control groups were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: TEM and NTA results demonstrated that UEVs were successfully extracted. Western blotting results confirmed that TSG101 was suitable as an internal reference for this study. Compared with the control group, the IgAN group showed positive expression of MASP2. MASP2 levels in the UEVs, determined by ELISA, showed significant differences between IgAN and control groups, which were significantly positively correlated with the level of urinary microalbumin. CONCLUSIONS: The level of MASP2 in UEVs was related to IgAN and shows promise as a biomarker for evaluating the severity of renal injury and prognosis of IgAN, thereby helping to elucidate the role of MASP2 in the mannan-binding lectin pathway.

Mucor fragilis as a novel source of the key pharmaceutical agents podophyllotoxin and kaempferol.

CONTEXT: Podophyllotoxin, a pharmaceutically important bioactive compound of Podophyllum sps. (Berberidaceae), is in great demand worldwide as an anticancer and antivirus drug precursor. However, the source of podophyllotoxin is very limited due to the endangered status of the Podophyllum plant. OBJECTIVE: The aim of this study was to isolate podophyllotoxin-producing endophytic fungi from Sinopodophyllum hexandrum (Royle) Ying (1979) (Berberidaceae) plants of the Taibai Mountains of China in order to obtain bioactive compounds. MATERIALS AND METHODS: The strains producing kaempferol and podophyllotoxin were screened by thin-layer chromatography (TLC) analysis. The presence of kaempferol and podophyllotoxin in extracts of these strains was further confirmed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analyses. RESULTS: Among six endophytic fungi isolated from the rhizomes of S. hexandrum, one strain was able to produce kaempferol. Another strain, named TW5, was able to produce both kaempferol and podophyllotoxin simultaneously according to the TLC, HPLC, and NMR results. The podophyllotoxin yield of TW5 was calculated to be 49.3 µg/g of mycelial dry weight after 7-d fermentation. Strain TW5 was identified morphologically and phylogenetically to be Mucor fragilis Fresen. (Mucoraceae). These results suggest that the podophyllotoxin-synthesizing ability is obtained by uptaking genes involved in the podophyllotoxin synthesis from the host plant into endophytic fungal genomes. CONCLUSION: Our results showed, for the first time, that the endophytic fungus M. fragilis is able to produce simultaneously the same two bioactive metabolites, podophyllotoxin and kaempferol, as its host plant. Furthermore, the relatively high podophyllotoxin yield obtained may improve the industrial production of podophyllotoxin, which may help protect this endangered plant.

[Expression and Clinical Significance of Eukaryotic Initiation Factor 4E in Newly Diagnosed Patients with Multiple Myeloma].

OBJECTIVE: To explore the expression and clinical significance of eukaryotic translation initiation factor 4E(eIF4E) in bone marrow samples of newly diagnosed multiple myeloma (NDMM) patients. METHODS: Immunohistochemical staining was used to analyze the expression of eIF4E protein in bone marrow biopsy samples from 75 NDMM patients and 25 patients with benign bone marrow disease. Clinical data were collected to analyze the correlation between eIF4E protein expression and clinical features and its prognostic significance in bone marrow samples of NDMM patients. RESULTS: The positive rate of eIF4E protein expression in bone marrow samples of NDMM patients was higher than that in patients with benign bone marrow disease (P<0.001). Positive expression of eIF4E protein was correlated with elevated serum lactate dehydrogenase in MM patients. Univariate and multivariate analysis showed that positive expression of eIF4E protein was significantly associated with shortened overall survival (OS) in NDMM patients, and was an independent risk factor affecting OS in NDMM patients. CONCLUSION: Positive expression of eIF4E protein is an independent poor prognostic factor for OS in NDMM patients.

rac-(1R,2S,6R,7R)-4-{[(1E)-(2-Chloro-phen-yl)methyl-idene]amino}-1-isopropyl-7-methyl-4-aza-tricyclo-[5.2.2.0]undec-8-ene-3,5-dione.

The title compound, C(21)H(23)ClN(2)O(2), was synthesized from N-amino-α-terpinene maleimide and 2-chloro-benzaldehyde. There are two independent mol-ecules in the asymmetric unit which are linked via an inter-molecular C-H⋯O hydrogen bond. The crystal studied was found to be a partial merohedral twin, with a 0.74 (7):0.26 (7) domain ratio.

Development of Time-Resolved Fluorescence Immunochromatographic Assays for Simultaneously Detecting Tylosin and Tilmicosin in Milk in Group-Screening Manner.

Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection.

Retroperitoneoscopic distal pancreatectomy: a new surgical approach.

INTRODUCTION: The traditional laparoscopic surgery is difficult to deal with the deep lesions of the body and tail of the pancreas, which may damage the visceral organs of the abdominal cavity and cause abdominal adhesion and other related complications. AIM: This paper introduces the operation procedure of retroperitoneoscopy in pancreatic surgery, and evaluates its feasibility in clinical application. MATERIAL AND METHODS: Retrospective analysis was performed on patients with retroperitoneal pancreatectomy in our hospital. The anatomical features of the fascia, surgical plane composition and surgical pathway of the fascia of the retroperitoneoscopic pancreatectomy were observed during the operation, and the surgical safety and feasibility were analyzed. The following parameters were evaluated: operation time, blood loss, pancreatic fistula, postoperative gastro-intestinal recovery, hospital stay. RESULTS: All 3 patients had a smooth operation and no serious complications occurred. During retroperitoneal laparoscopic pancreatectomy, there is a vascularized plane between the posterior fascia of the pancreas and the prerenal fascia, which can avoid injury of the visceral organs and retroperitoneal vessels. The anterior renal fascia should be used as the posterior boundary of the safe separation plane. CONCLUSIONS: The surgical plane based on the anatomy of the fascia and interstitial dissection is the theoretical basis of modern surgery, which is safe, fast and effective. The inter-prerenal fascia plane is the correct and safe anatomical plane of posterior laparoscopic surgery.

Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples.

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples.

[Synthesis of gastrodin by microbial transformation].

AIM: To synthesize gastrodin via biotransformation of microorganism using p-hydroxybenzaldehyde as substrate. METHODS: Using resting cell techniques with TLC and HPLC analysis, a strain of Rhizopus chinensis Staito AS3. 1165 which has the capability of biotransforming p-hydroxybenzaldehyde into gastrodin was screened from 50 strains of microorganisms. Preparative scale biotransformation was carried out under the optimal transforming condition. The product was obtained by chromatography, and characterized on the basis of its physical and spectral data. RESULTS: The product was identified as gastrodin on the basis of its 1H NMR, 13C NMR and EI-MS spectral data. CONCLUSION: The fact that gastrodin could be synthesized by microbial transformation implies a new approach to gastrodin production. As a result, this research will be of a great economic and social value.

[Expression of MicroRNA-26a and Its Target Gene CDK6 in Extra-nodal NK/T-Cell Lymphoma].

OBJECTIVE: To explore the expression level of microRNA-26a and its target gene CDK6 in extranodal NK/T-cell lymphoma (ENKTCL) and their possible role in genesis and development of ENKTCL. METHODS: Real time fluorescent quantitative PCR was used to detect the expression level of miR-26a in tissue of 15 patients with ENKTCL and 10 samples of normal NK cells. Maxvision immunohistochemistry technique was used to detect the expression level of CDK6 and miR-26a in tissue of 20 ENKTCL cases, 10 cases of proliferative lymphadenitis and 10 samples of normal lymph node, respectively. The possible role of miR-26a and its target gene CDK6 in genesis and development of ENKTCL were analyzed according to the clinical features of ENKTCL patients. RESULTS: The expression of miR-26a was significantly lower in ENKTCL than that in normal NK cells. The expression of CDK6 was significantly higher in ENKTCL group than that in group proliferative lymphadenitis and normal lymph node. Correlation analysis showed that there was significant negative correlation between miR-26a expression and CDK6 expression (r = -0.54, P = 0.04). Meanwhile, there were no correlation of miR-26a expression with age, sex, Ann Arbor stage, LDH level, B symptoms and IPI. Although, there were no correlation of CDK6 expression with age, sex, LDH level and B symptoms, there were positive correlation of CDK6 expression with Ann Arbor stage and IPI. CONCLUSION: that abnormal expression of miR-26a may participate in genesis and development of ENKTCL through regulating the expression of its target gene CDK6.

Evaluation of fibroblast growth factor signaling during lens fiber cell differentiation.

PURPOSE: Previous studies have implicated members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as stimulators of lens fiber cell differentiation in rodent and chicken embryo lenses, respectively. In the present study, the role of FGFs in fiber cell differentiation and epithelial cell proliferation in chicken embryos was examined. METHODS: Lenses were injected on embryonic day (E)3 with replication-defective retroviruses that express full-length or truncated FGF receptor (FGFR)-1 or a secreted form of FGF1. Lens epithelial explants were cultured in defined medium or medium supplemented with FGFs or vitreous humor, in the presence or absence of the FGF receptor antagonist SU5402. Explants were also cultured in vitreous humor that had been depleted of heparin-binding growth factors. Cell elongation was measured optically and protein accumulation by densitometry and Western blot analysis. RESULTS: Lens fiber cell differentiation was not inhibited in cells infected with virus expressing truncated FGFR1. Epithelial cells infected with virus encoding a secreted form of FGF1 did not differentiate into ectopic fiber cells. Viral transduction of FGFR1, truncated FGFR1, or FGF1 did not appreciably alter the proliferation of lens epithelial cells. Bovine vitreous humor stimulated chicken embryo lens epithelial cells to elongate and express markers of lens fiber cell differentiation. Bovine vitreous humor, but not FGF2, protected lens epithelial cells from apoptosis. Depleting vitreous humor of heparin-binding growth factors or treatment of lens cells with SU5402 did not inhibit the initial, rapid phase of lens cell elongation. Both treatments, used separately or together, reduced but did not prevent the expression of later markers of fiber cell differentiation. CONCLUSIONS: Fiber differentiation factors that are not members of the FGF family are present in chicken and mammalian vitreous humor. The factors that stimulate fiber cell differentiation in avian and mammalian eyes are similar.

[Preparation of hen egg yolk immunoglobulin and partial plasma high abundant protein depletion by prepared chicken immunoglobulin coupled on GoldMag carrier].

AIM: To prepare specific egg yolk immunoglobulin (IgY) against HSA and IgG and to make HSA and IgG depletion research in human plasma by binding it to the surface of GoldMag. METHODS: Laying Roman hens immunized with different antigens, such as HSA, IgG and the mixtures of these proteins were used in the experiments. The best condition of isolating the IgY antibody was studied. The titer and purity of the antibody were examined by indirect ELISA and SDS-PAGE respectively. Then the specific IgY was bound to the surface of GoldMag for HSA and IgG depletion research. RESULTS: 60-120 days after immunization, the titer of the antibody in the plasma against different antigens reached 1:15 000-1:25 000; while the titer in the egg reached 1:10 000-1:25 000. The purity was over 98%. Most of HSA and IgG were depleted from human plasma by IgY connected to GoldMag. CONCLUSION: HSA and IgG can be used as antigens to immunize laying hens to produce IgY with high titer and purity. IgY which is bound to the GoldMag carrier can be used to deplete of the relevant HSA and IgG from human plasma effectively. As a new method of studying proteome of plasma, it has some practical applications.