Establishment of an indirect ELISA-based method involving the use of a multiepitope recombinant S protein to detect antibodies against canine coronavirus.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91-135 aa, S1 gene; 377-434 aa, S2 gene; 647-671 aa, S3 gene; 951-971 aa, S4 gene; 207-227 aa) and two T-cell epitopes in tandem: T-E1-E2-E3-E4-T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

Lactobacillus hegangensis sp. nov., Lactobacillus suibinensis sp. nov., Lactobacillus daqingensis sp. nov., Lactobacillus yichunensis sp. nov., Lactobacillus mulanensis sp. nov., Lactobacillus achengensis sp. nov., Lactobacillus wuchangensis sp. nov., Lactobacillus gannanensis sp. nov., Lactobacillus binensis sp. nov. and Lactobacillus angrenensis sp. nov., isolated from Chinese traditional pickle and yogurt.

Fourteen Gram-stain-positive bacterial strains were isolated from Chinese traditional pickle and yogurt. The strains were characterized using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, determination of average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH) and an analysis of phenotypic features. The data demonstrated that the 14 strains represented ten novel species belonging to the genus Lactobacillus, strains 73-4T, 247-3T, 143-4(a)T, 33-1T, 143-6T, 247-4T, 17-4T, 143-1T, 735-2T and M1530-1T were designated as the type strains. Strains 73-4T and 247-3T were phylogenetically related to the type strains of Lactobacillus camelliae and Lactobacillus jixianensis, having 97.0-98.9 % 16S rRNA gene sequence similarities, 83.9-87.2 % pheS gene sequence similarities and 86.8-93.3 % rpoA gene sequence similarities. Strains 143-4(a)T and 33-1T were phylogenetically related to the type strains of Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus casei, having 93.6-96.5 % 16S rRNA gene sequence similarities, 73.9-77.2 % pheS gene sequence similarities and 76.1-77.6 % rpoA gene sequence similarities. Strains 143-6T, 247-4T, 17-4T and 143-1T were phylogenetically related to the type strains of Lactobacillus concavus, Lactobacillus dextrinicus and Lactobacillus bayanensis, exhibiting 95.5-99.9 % 16S rRNA gene sequence similarities, 76.5-83.1 % pheS gene sequence similarities and 83.6-98.3 % rpoA gene sequence similarities. Strain 735-2T was phylogenetically related to the type strains of Lactobacillus zhaoyuanensis, Lactobacillus jiayinensis and Lactobacillus coryniformis, having 98.2-99.1 % 16S rRNA gene sequence similarities, 82.8-84.1 % pheS gene sequence similarities and 93.0-93.9 % rpoA gene sequence similarities. Strain M1530-1T was phylogenetically related to the type strains of Lactobacillus suantsaiihabitans and Lactobacillus brevis, having 99.5 and 99.0 % 16S rRNA gene sequence similarities, 90.3 and 81.7 % pheS gene sequence similarities and 97.7 and 91.1 % rpoA gene sequence similarities. The ANI and isDDH values between strains 73-4T, 247-3T, 143-4(a)T, 33-1T, 143-6T, 247-4T, 17-4T, 143-1T, 735-2T, M1530-1T and type strains of phylogenetically related species were less than 86.8 % and 33.9 % respectively, confirming that they represent ten novel species within the genus Lactobacillus. Based upon the data of polyphasic characterization obtained in the present study, ten novel species, Lactobacillus hegangensis sp. nov., Lactobacillus suibinensis sp. nov., Lactobacillus daqingensis sp. nov., Lactobacillus yichunensis sp. nov., Lactobacillus mulanensis sp. nov., Lactobacillus achengensis sp. nov., Lactobacillus wuchangensis sp. nov., Lactobacillus gannanensis sp. nov., Lactobacillus binensis sp. nov. and Lactobacillus angrenensis sp. nov., are proposed and the type strains are 73-4T (=NCIMB 15177T=CCM 8912T=CCTCC AB 2018407T), 247-3T (=NCIMB 15176T=JCM 33275T), 143-4(a)T (=NCIMB 15173T=CCM 8948T=JCM 33273T=CCTCC AB 2018390T), 33-1T (=NCIMB 15169T=CCM 8947T=JCM 33272T=CCTCC AB 2018405T), 143-6T (=NCIMB 15162T=CCM 8951T=JCM 33274T=CCTCC AB 2018411T), 247-4T (=NCIMB 15155T=CCM 8897T=LMG 31059T=CCTCC AB 2018410T), 17-4T (=NCIMB 15161T=CCM 8946T=JCM 33271T=CCTCC AB 2018406T), 143-1T (=NCIMB 15157T=CCM 8937T=CCTCC AB 2018409T), 735-2T (=NCIMB 15190T=CCM 8925T=LMG 31186T) and M1530-1T (=NCIMB 15150T=CCM 8893T=LMG 31046T=CCTCC AB 2018402T), respectively.

Heparin-binding protein measurement improves the prediction of myocardial injury-related cardiogenic shock.

BACKGROUND: Heparin-binding protein (HBP), a potent inducer of increased vascular permeability, is a potentially useful biomarker for predicting outcomes in patients with postoperative myocardial injury-related cardiogenic shock (MIRCS). We aimed to evaluate and validate HBP as a prognostic biomarker for postoperative MIRCS. METHODS: We performed a case-control study in 792 patients undergoing cardiac surgery from January 1, 2016, to August 1, 2019, including 172 patients with postoperative MIRCS and 620 age- and sex-matched controls. The association between HBP and MIRCS was determined by multivariate logistic regression analysis. Receiver operating characteristic curves (ROCs) with area under the curve (AUC) were performed to calculate the cut-off value, sensitivity and specificity. The association between HBP and cardiac troponin T (cTnT) was determined by multivariable linear regression analysis. Blood samples were drawn from the coronary sinus and arterial line of the cardiopulmonary bypass (CPB) before aortic cross-clamping (time point 1) and 5 min after aortic declamping (time point 2). RESULTS: Before aortic cross-clamping, coronary sinus HBP (HBPCS1) showed no differences between the two groups. However, after declamping, the MIRCS group had a significantly higher sinus HBP level (HBPCS2) than did the control group. HBPCS2 predicted MIRCS with an AUC of 0.85 (95% CI: 0.81-0.89, cut-off: 220 ng/ml, sensitivity: 92% and specificity: 70%). After adjusting for confounding factors, we found that HBP was an independent risk factor for MIRCS (OR: 7.65, 95% CI: 4.86-12.06, P < 0.01) and was positively associated with cTnT (ß > 0, P < 0.01). CONCLUSIONS: Elevated levels of coronary sinus HBP were useful biomarkers for predicting MIRCS after cardiac surgery.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

Lactobacillus jixianensis sp. nov., Lactobacillus baoqingensis sp. nov., Lactobacillus jiayinensis sp. nov., Lactobacillus zhaoyuanensis sp. nov., Lactobacillus lindianensis sp. nov., Lactobacillus huananensis sp. nov., Lactobacillus tangyuanensis sp. nov., Lactobacillus fuyuanensis sp. nov., Lactobacillus tongjiangensis sp. nov., Lactobacillus fujinensis sp. nov. and Lactobacillus mulengensis sp. nov., isolated from Chinese traditional pickle.

Thirty Gram-stain-positive bacterial strains were isolated from Chinese traditional pickle. The strains were characterized using a polyphasic taxonomic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, determination of DNA G+C content, determination of average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), determination of average amino acid identity (AAI) and an analysis of phenotypic features. The data demonstrated that the 30 strains represented 11 novel species belonging to the genus Lactobacillus, strains 159-4T, 47-3T, 257-1T, 187-3T, 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T and 112-3T were designated as the type strains. Strains 159-4T and 47-3T were related to the type strains of Lactobacillus porcinae, Lactobacillus manihotivorans, Lactobacillus nasuensis, Lactobacillus camelliae, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus sharpeae and Lactobacillus songhuajiangensis, having 92.0-98.9 % 16S rRNA gene sequence similarities, 58.1-84.7 % pheS gene sequence similarities and 61.4-90.3 % rpoA gene sequence similarities. Strains 257-1T and 187-3T were related to the type strains of Lactobacillus coryniformis, Lactobacillus iwatensis, Lactobacillus backii, Lactobacillus rennini and Lactobacillus bifermentans, having 93.5-99.3 % 16S rRNA gene sequence similarities, 67.7-81.8 % pheS gene sequence similarities and 77.0-96.2 % rpoA gene sequence similarities. Strains 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T and 112-3T were closely related to the type strains of Lactobacillus paucivorans, Lactobacillus brevis, Lactobacillus hammesii, Lactobacillus senmaizukei, Lactobacillus parabrevis, Lactobacillus yonginensis, Lactobacillus koreensis and Lactobacillus cerevisiae, having 95.6-99.9 % 16S rRNA gene sequence similarities, less than 93.9 % pheS gene sequence similarities and 87.0-99.7 % rpoA gene sequence similarities. ANI, isDDH and AAI values between strains 159-4T, 47-3T, 257-1T, 187-3T, 220-4T, 151-2BT, 137-3T, 244-4T, 218-10T, 218-6T, 112-3T and type strains of phylogenetically related species were less than 92.7, 48.4 and 96.6 %, respectively, confirming that they represent 11 novel species within the genus Lactobacillus. Based upon the data of polyphasic characterization obtained in the present study, eleven novel species, Lactobacillusjixianensis sp. nov., Lactobacillusbaoqingensis sp. nov., Lactobacillusjiayinensis sp. nov., Lactobacilluszhaoyuanensis sp. nov., Lactobacilluslindianensis sp. nov., Lactobacillushuananensis sp. nov., Lactobacillustangyuanensis sp. nov., Lactobacillusfuyuanensis sp. nov., Lactobacillustongjiangensis sp. nov., Lactobacillusfujinensis sp. nov. and Lactobacillusmulengensis sp. nov., are proposed and the type strains are 159-4T (=NCIMB 15175T=CCM 8911T), 47-3T (=NCIMB 15165T=CCM 8903T=LMG 31064T), 257-1T (=NCIMB 15166T=CCM 8904T=LMG 31065T), 187-3T (=NCIMB 15172T =CCM 8910T), 220-4T (=NCIMB 15163T =CCM 8902T=KCTC 21136T), 151-2BT (=NCIMB 15164T=CCM 8913T=KCTC 21129T=LMG 31063T), 137-3T (=NCIMB 15170T=CCM 8907T=KCTC 21125T=LMG 31053T), 244-4T (=NCIMB 15168T=CCM 8906T=KCTC 21137T=LMG 31052T), 218-10T (=NCIMB 15167T=CCM 8905T=KCTC 21135T =LMG 31055T), 218-6T (=NCIMB 15171T=CCM 8908T=KCTC 21134T =LMG 31067T) and 112-3T (=NCIMB 15174T=CCM 8909T=KCTC 21123T=LMG 31049T), respectively.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979 946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

Misplacement of Tunneled Hemodialysis Catheter into Azygos Vein: Left or Right Jugular Insertion Has Similar Susceptibility.

BACKGROUND: Central venous catheter (CVC) is commonly used to provide access for hemodialysis (HD) when arteriovenous access is not available. The misplacement of CVC into azygos vein (AV) is a rare but a potential serious complication. Previous reports communicated the opinion that left-sided catheterization predisposed to AV misplacement, but these reports concentrated on peripherally inserted CVCs, placed for indications rather than HD. Unintended AV misplacement of HD catheters (HDCs) has not been well studied. We seek to investigate factors associated with inadvertent AV miscannulation during HDC placement. METHODS: We are to present a case of unintentional misplacement of a tunneled HD catheter (tHDC) into the azygos arch from right internal jugular vein (RIJV) despite real-time fluoroscopy guidance. Additionally, we have undertaken a systematic literature search in Pubmed to study the anatomical and other factors related to unintended AV misposition in HD setting. RESULTS: From 2005 to August 31, 2018, a total of 11 articles containing 16 cases of misplacement of HDCs into AV were identified. Of the 17 cases of unintentional AV misposition including ours, the majority of the misguided HDCs (94.1%, 16/17) were tHDCs and only 1 case was related to a temporary (non-tunneled) catheter. Most catheter misplacements (88.2%, 15/17) were performed without real-time radiological guidance. The reported incidence of inadvertent AV cannulation from different institutions varied between 0.6% and 3.8%. Among the 16 misplaced tHDCs, the rates of AV misposition that arose from RIJV and left internal jugular vein (LIJV) insertion are even at 50%. CONCLUSIONS: Based upon anatomical and case studies, we have found that AV may join posterior aspect of superior vena cava at different directions and levels. Hence, this might explain why AV misplacement might occur whether an HDC is inserted from the LIJV or RIJV approach. By raising the awareness of this potential complication and how we may minimize it, we hope to reduce the future complication of AV misposition.

Effects of dietary inclusion levels of palm oil on growth performance, antioxidative status and serum cytokines of broiler chickens.

This study was conducted to investigate the effects of dietary palm oil inclusion levels on growth performance, serum antioxidative status and cytokines in Sanhuang broiler chickens. A total of 208 one-day-old female Sanhuang chickens were randomly allocated to four treatment groups with four replicates per group and of 13 birds each for a 42-day feeding trial. Dietary treatments were basal diet without palm oil (control) and diets containing 2%, 4% or 6% palm oil. During days 22 to 42 and the overall experimental period, chickens fed diets supplemented with 4% and 6% palm oil had a lower average daily feed intake (ADFI) (p = 0.019) and feed to gain ratio (F/G) (p = 0.031). Average daily gain (ADG) tended to be higher (p = 0.089) for palm oil inclusion treatments compared with the control treatment from days 1 to 42. Dietary 2% and 4% palm oil treatments significantly lowed serum malondialdehyde concentration on day 21 when compared to the control and 6% palm oil treatments (p = 0.027). Dietary 6% palm oil treatment significantly reduced the catalase (CAT) activity (p = 0.018) and total antioxidant capacity (T-AOC) (p = 0.027) in serum in comparison with the control treatment on day 21. No significant differences were observed in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in serum among dietary treatments on day 21. There were no significant differences in T-AOC, CAT, SOD and GSH-Px activities among dietary treatments on day 42. Dietary inclusion of 4% and 6% palm oil significantly increased (p = 0.01) serum interleukin-1ß (IL-1ß) content, but improved transforming growth factor-ß (TGF-ß) content in serum on both days 21 and 42 (p = 0.032 and 0.022 respectively) as well as IL-10 content on day 42 (p = 0.022) when compared with the control treatment. Furthermore, dietary inclusion of 6% palm oil significantly reduced tumour necrosis factor-α (TNF-α) content on days 21 and 42 (p = 0.021 and 0.001 respectively) compared with the control treatment. These results suggest that dietary inclusion of 4% and 6% palm oil could improve the growth performance, modulate the balance between pro- and anti-inflammatory cytokines, and show limited effect on antioxidative status in Sanhuang broiler chickens.

Inclusion of Konjac Flour in the Gestation Diet Changes the Gut Microbiota, Alleviates Oxidative Stress, and Improves Insulin Sensitivity in Sows.

UNLABELLED: Although dietary fibers contribute to health and physiology primarily via the fermentative actions of the gut microbiota of the hosts, few studies have focused on how these interactions influence the metabolic status of sows. Here, the effects of inclusion of konjac flour (KF) in a gestation diet on oxidative stress status, insulin sensitivity, and gut microbiota were investigated to elucidate the correlation between the microbiota and metabolic changes in sows. Sows were assigned to either control or 2.2% KF dietary treatment during gestation. The gut microbiota population in sows during gestation and lactation was assessed by 16S rRNA gene sequencing. The oxidative stress parameters, homeostasis model assessment (HOMA) values, and fatty acids in the blood of sows were also assessed. Compared to the control diet group, KF significantly reduced the serum levels of reactive oxygen species (ROS) and 8-hydroxy-deoxyguanosine (8-OHdG) but increased the serum concentrations of glutathione peroxidase (GSH-Px) in sows on day 1 in lactation. Additionally, sows in the KF group had a lower HOMA insulin resistance value but a higher HOMA insulin sensitivity (HOMA-IS) value. KF induced changes in the gut microbial composition at the phylum and genus levels. The increased relative abundances of Akkermansia and Roseburia in the KF group were positively correlated with the HOMA-IS. Overall, dietary KF alleviated oxidative stress and improved insulin sensitivity of sows, and the changes in the gut microbiota in response to KF may have been correlated with the host metabolism response. IMPORTANCE: To date, the effect of dietary fiber on metabolism responses and gut microbiota in sows has not been investigated. Here, KF supplementation of a gestation diet in sows was found to alleviate oxidative stress and to improve insulin sensitivity. Pyrosequencing analysis revealed that KF treatment induces changes in the gut microbiota composition at the phylum and genus levels. Moreover, the changes of gut microbiota in response to KF may be correlated with the host metabolism response.

[A semimicroquality evaluation method on Panax notoginseng and its application in analysis of continuous cropping obstacles research samples].

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Elevated Plasma P-Selectin Autoantibodies in Primary Sjögren Syndrome Patients with Thrombocytopenia.

BACKGROUND Primary Sjögren's syndrome (pSS) is one of the most common chronic systemic autoimmune diseases, and thrombocytopenia is one of the hematological manifestations of pSS. When platelet and endothelial cells are activated, P-selectin is expressed on the cell surface. This study aimed to investigate the role of P-selectin autoantibodies in the pathogenesis of thrombocytopenia in pSS. MATERIAL AND METHODS P-selectin autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) in 38 pSS patients without thrombocytopenia and 32 pSS patients with thrombocytopenia, 32 idiopathic thrombocytopenic purpura (ITP) patients, and 35 healthy controls. RESULTS The plasma P-selectin autoantibodies (A490) in ITP patients and pSS patients with/without thrombocytopenia were significantly higher than those in healthy controls, but there were no significant differences between ITP patients and pSS patients with thrombocytopenia. The positive rate of P-selectin autoantibodies in pSS patients with thrombocytopenia was significantly higher than that in ITP patients. The platelet count was lower in P-selectin autoantibodies-positive patients, while among pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients than in P-selectin autoantibodies-negative patients. In ITP patients and pSS patients with thrombocytopenia, the platelet count was lower in P-selectin autoantibodies-positive patients. CONCLUSIONS Elevated plasma P-selectin autoantibodies may play a role in the pathogenesis of thrombocytopenia in pSS patients.

[Wild resources survey of Marsdenia tenacissima in Honghe, Yunnan].

To ascertain current situation of wild Marsdenia tenacissima resources in Honghe, Yunnan province, the distribution, habitat characteristic and resources reserves of M. tenacissima were surveyed based on interviews and investigation. The results showed that M. tenacissima was found in 7 counties such as Jinping, Mengzi etc, and distributed mainly on the mountainsides from 800 m to 1 200 m. And distribution was affected by many factors, such as light, heat, topography, soil, and vegetation. M. tenacissima grew well in distribution areas. M. tenacissima had averagely a weight of 2.8 kg per plant. Resources reserve of M. tenacissima in Honghe was estimated to 1 300 tons by now but it reduced rapidly in resent years, the wild resources reserve may not meet demand of market. Resources protection and wildlife tending would be conducted to deal with increasing medication requirements.

Matrine inhibits disturbed flow-enhanced migration via downregulation of ERK1/2-MLCK signaling vascular smooth muscle cells.

BACKGROUND: To investigate the effects of matrine on the vascular smooth muscle cell (VSMC) migration modulated by disturbed flow and their underlying molecular mechanisms in vitro. METHODS: Isolated rat aortic VSMCs were grown to confluence on 20- × 80-mm fibronectin-coated glass cover slides, and then, denuded zones were made at the position calculated to be the oscillating flow-reattachment zone and also in the downstream laminar flow region. VSMCs were treated with different doses of matrine (0, 10, 20, 30, and 40 mg/L), or PD98059 (30 µM), ML-7 (10 µM) combined with matrine (40 mg/L) for 30 minutes before and during the experiments. Then, the wounded monolayers were kept under static conditions or were subjected to laminar or disturbed flow for 21 hours or 10 hours. The VSMC migration was assessed by microscopic images. The extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) proteins were determined by Western blot. RESULTS: Disturbed flow significantly increased phosphorylation of ERK1/2. Selective inhibition of ERK1/2 phosphorylation by inhibitor PD98059 and matrine significantly suppressed VSMC migration under disturbed flow. Disturbed flow significantly enhanced phosphorylation of MLCK, whereas both matrine and PD98059 inhibited the phosphorylation of MLCK under disturbed flow. The complete inhibition of MLCK phosphorylation using the selective MLCK inhibitor ML-7 significantly inhibited VSMC migration under disturbed flow. CONCLUSION: Matrine inhibits VSMC migration under disturbed flow, in part, by downregulation of ERK1/2-MLCK signaling pathway.

Ethyl 7-methyl-2-((1-methyl-1H-pyrrol-2-yl)methyl-ene)-3-oxo-5-phenyl-3,5-dihydro-2H-thia-zolo[3,2-a]pyrimidine-6-carboxyl-ate.

In the structure of the title compound, C(22)H(21)N(3)O(3)S, the thia-zole ring forms dihedral angles of 88.83 (7) and 9.39 (9)°, respectively, with the benzene and pyrrole rings. The dihydro-pyrimidine ring adopts a flattened boat conformation. The olefinic double bond is in a Z conformation.

[Effects of intercropping and nitrogen application on soil microbial metabolic functional diversity in maize cropping soil.] / çŽ‰ç±³é—´ä½œå’Œæ–½æ°®å¯¹åœŸå£¤å¾®ç”Ÿç‰©ä»£è°¢åŠŸèƒ½å¤šæ ·æ€§çš„å½±å“.

Soil microorganism is an important indicator of soil health and plays a critical role in biogeochemical processes. We collected soil samples from a multi-year in-situ field experiment with two cropping modes (maize monocropping and maize-potato intercropping) subjected to four nitrogen (N) levels (N0, 0 kg·hm-2; N125, 125 kg·hm-2; N250, 250 kg·hm-2; N375, 375 kg·hm-2). By using the Biolog-ECO microplate method, soil microbial metabolic activity, diversity and utilization of six carbon groups were analyzed. The results showed that N application significantly increased the average well color development (AWCD) values, Simpson and Shannon indices, with the highest value at N250 in mono- and inter-cropped soils. Moreover, N application promoted the utilization of amino acids, amines and phenolic compounds, but decreased the utilization of polymers. Compared with monocropping at the same N level, intercropping improved the AWCD values, Simpson and Shannon indices, with a maximal improvement at N250. Intercropping and nitrogen application significantly affected the metabolic activities of the six carbon-source groups. In addition, intercropping improved the utilization of labile carbon sources, such as amino acids and carbohydrates. Results of the redundancy analysis and linear regression showed that intercropping and N application elevated AWCD values by increasing soil temperature, water content, and soil organic carbon content. Therefore, the changes in soil physicochemical properties after intercropping were the main reason for the enhancement of microbial metabolic activity under N application.

Nanodevices for deep cartilage penetration.

Osteoarthritis (OA) is a degenerative joint disease and is the main cause of chronic pain and functional disability in adults. Articular cartilage is a hydrated soft tissue that is composed of normally quiescent chondrocytes at a low density, a dense network of collagen fibrils with a pore size of 60-200 nm, and aggrecan proteoglycans with high-density negative charge. Although certain drugs, nucleic acids, and proteins have the potential to slow the progression of OA and restore the joints, these treatments have not been clinically applied owing to the lack of an effective delivery system capable of breaking through the cartilage barrier. Recently, the development of nanotechnology for delivery systems renders new ideas and treatment methods viable in overcoming the limited penetration. In this review, we focus on current research on such applications of nanotechnology, including exosomes, protein-based cationic nanocarriers, cationic liposomes/solid lipid nanoparticles, amino acid-based nanocarriers, polyamide derivatives-based nanocarriers, manganese dioxide, and carbon nanotubes. Exosomes are the smallest known nanoscale extracellular vesicles, and they can quickly deliver nucleic acids or proteins to the required depth. Through electrostatic interactions, nanocarriers with appropriate balance in cationic property and particle size have a strong ability to penetrate cartilage. Although substantial preclinical evidence has been obtained, further optimization is necessary for clinical transformation. STATEMENT OF SIGNIFICANCE: The dense cartilage matrix with high-negative charge was associated with reduced therapeutic effect in osteoarthritis patients with deep pathological changes. However, a systematic review in nanodevices for deep cartilage penetration is still lacking. Current approaches to assure penetration of nanosystems into the depth of cartilage were reviewed, including nanoscale extracellular vesicles from different cell lines and nanocarriers with appropriate balance in cationic property and size particle. Moreover, nanodevices entering clinical trials and further optimization were also discussed, providing important guiding significance to future research.

Protection of endothelial cells, inhibition of neointimal hyperplasia by ß-elemene in an injured artery.

AIMS: It is generally accepted that the oxidative stress and the proliferative activity of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of neointimal hyperplasia after vascular injury. Although ß-elemene (ß-1-methyl-1-vinyl-2, 4-diisopropenyl-cyclohexane) has been used as an antitumour drug, its therapeutic effect on vascular diseases has not yet been determined. In this study, we investigated whether ß-elemene could inhibit oxidative damage of vascular endothelial cells, suppress VSMCs growth and prevent neointimal hyperplasia. METHODS AND RESULTS: ß-elemene can increase the survival rate of human umbilical vein endothelial cells in vitro. By measuring the malondialdehyde content, total antioxidant capacity, superoxide dismutase activity, catalase activity, glutathione peroxidase activity and nitric oxide levels, we assessed the protective effect of ß-elemene in the vascular endothelium model against oxidant-induced injury. Μoreover, ß-elemene inhibited cell proliferation and induced apoptosis in cultured VSMCs. In a flow culture system, ß-elemene reduced the migration distance of VSMCs. By transwell migration assay, ß-elemene was found to reduce the migration cell number of VSMCs, but not affect the HUVECs migration. In a rat carotid artery balloon injury model, administration of ß-elemene significantly reduced the ratio of intimal area to medial area and neointima formation. CONCLUSIONS: Our results indicate that ß-elemene is effective in protecting the endothelial cells from injury induced by hydrogen peroxide in vitro, inhibiting smooth muscle cell proliferation/migration and inhibiting neointima formation in vivo after vascular injury.

[Regulatory effects of glutamine on Toll-like receptor 4 in neonatal rats with necrotizing enterocolitis].

OBJECTIVE: To study the expression of Toll-like receptor 4 (TLR-4) and caspase-3 in the intestine of neonatal rats with necrotizing enterocolitis (NEC), and explore the protective effects and possible regulatory mechanisms of glutamine (Gln) in NEC. METHODS: Sixty premature rats were randomly divided into three groups (n=20 each): control, NEC model and Gln intervention group. NEC model was prepared by formula feeding, hypoxia and cold stress. The Gln intervention group was also subjected to hypoxia and cold stress but was fed with formula containing Gln (0.3 g/kg). Two days later, the rats were sacrificed and the intestine tissues were obtained. The histological changes of ileal tissues were observed by hemetoxylin and eosin staining. The expression of caspase-3 and TLR-4 protein in the jejunum, ileum and colon were detected by inmunohistochemistry. The expression of TLR-4 mRNA in the jejunum, ileum and colon were detected by RT-PCR. RESULTS: Compared with the control group, the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA in the NEC model group increased significantly (P<0.01). Gln intervention decreased significantly the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA compared with the NEC model group (P<0.05). CONCLUSIONS: TLR-4 might be involved in the pathogenesis of NEC. Gln may provide protective effects on intestine possibly through reducing the TLR-4 expression and then decreasing the apoptosis of intestinal epithelial cells.

Matrine inhibits vascular smooth muscle cell proliferation by modulating the expression of cell cycle regulatory genes.

AIM: To investigate the effect of matrine on proliferation of vascular smooth muscle cells (VSMCs) and elucidate the underlying mechanisms. METHODS: Rat aortic VSMCs were cultured in medium supplemented with 10% fetal bovine serum and treated with various concentrations (0, 5, 10, 15, and 20 mg/L) of matrine for 72 h. VSMCs proliferation and cell cycle profiling were assessed using a methylene blue incorporation assay and flow cytometry, respectively. The underlying protein signaling mechanisms were determined using Western blot analysis of the expression levels of cell cycle regulatory genes, including p53, p21, p27, cyclin D1, cyclin E, cyclin-dependent kinase 2 and 4 (cdk2, cdk4), and phosphorylated Rb. The involvement of p21 and p27 pathways was further determined using small interfering RNA (siRNA) knockdown. RESULTS: Matrine inhibited VSMC proliferation in a dose-dependent manner by promoting G(1) arrest. The G(1) arrest was accompanied by up-regulation of p53 and p21 protein levels, and down-regulation of cyclin D1/cdk4, cyclin E/cdk2 and phosphorylated Rb protein levels. Matrine did not affect p27 expression. Furthermore, the anti-proliferative effect of matrine was abolished by silencing of p21, but not by silencing of p27. CONCLUSION: Our data indicate that matrine has an inhibitory effect on VSMC proliferation via up-regulation of the p53/p21 signaling pathway and modulation of other cell cycle regulatory genes.

[Predictive value of three surgical scoring systems for estimation of life expectancy in patients with extradural spinal metastasis].

OBJECTIVE: To evaluate the predictive values of Tokuhashi score, revised Tokuhashi score and Tomita score systems for life expectancy and treatment options in patients with spinal metastasis. METHODS: From February 1996 to January 2009, spinal operations in 104 cases with spinal metastasis were performed in our hospital. There were 65 males and 39 females, with an average of 53.4 years (median 52.5 years). To calculate AUC (area under the curve) values of Receiver Operating Characteristic (ROC) curves of three scores, and to analyze the accuracy of prediction of life expectancy. To compare the actual survival time with the expected survival time of the three scores by Kaplan-Meier method. Spearman correlation analysis was performed between the survival time and three scoring systems. RESULTS: All cases were followed-up with an average duration of 10.9 months, and 77 patients died. AUC analysis of ROC curves showed that the difference of the accuracy of the three scores was not significant. AUC in all groups of Tokuhashi Score was low, with a poor diagnostic accuracy. In the "died within 3 months" and "died within 6 months" groups of revised Tokuhashi score, the accuracy was low, while high in the other two groups. The AUC values of Tomita score in "died within 6 months" and "died within 24 months" were high, with a great diagnostic accuracy while the other two groups were low with a low diagnostic accuracy. Kaplan-Meier survival curve analysis showed that the actual survival time in all three scores was not entirely consistent with the expected survival time. Tokuhashi score and revised score were positively correlated with the survival time while that of Tomita score was negative. CONCLUSION: All the three prognosis scores in patients with spinal metastasis were closely related with survival time. The combination of Tokuhashi score and Tomita score may be applied to better predict postoperative survival prognosis and guide the surgical options for patients with spinal metastasis.