A novel pectate lyase with high specific activity from Bacillus sp. B58-2: Gene cloning, heterologous expression and use in ramie degumming.

Pectinase plays a crucial role in ramie degumming. A gene encoding a putative pectate lyase from Bacillus sp. strain B58-2 was cloned and heterologously expressed in Escherichia coli. The amplified gene BvelPL1 encoded a mature protein of 400 amino acids. BvelPL1 shared the highest amino acid sequence identity (78.75%) with the enzymatically characterized pectate lyase Pel from Bacillus subtilis strain RCK (GenBank: AFH66771.1). The purified recombinant enzyme rBvelPL1-Ec exhibited a maximum specific activity of 2433.26 U/mg at pH 8.5 and 50 °C towards polygalacturonic acid. This specific activity was higher than that of most reported pectate lyases. Remarkably, the enzymatic activity of rBvelPL1-Ec increased by 23.28 times in the presence of 0.4 mM calcium ion. The effect of calcium ion on promoting the enzymatic activity of rBvelPL1-Ec was greater than that for all reported pectate lyases. After degumming with rBvelPL1-Ec, a weight loss of 21.27 ± 1.17% of circled ramie fibers was obtained, and the surfaces of the ramie fibers became smoother. Moreover, a weight loss of 30.47 ± 0.46% was obtained through enzymatic treated and subsequent NaOH treated circled ramie fibers. The excellent performance in degumming suggests that rBvelPL1-Ec may serve as a promising biocatalyst in the textile industry.

Gene cloning, expression, and characterization of two endo-xylanases from Bacillus velezensis and Streptomyces rochei, and their application in xylooligosaccharide production.

Endo-xylanase hydrolyzing xylan in cellulosic residues releasing xylobiose as the major product at neutral pH are desirable in the substitute sweeteners industry. In this study, two endo-xylanases were obtained from Streptomyces rochei and Bacillus velezensis. SrocXyn10 showed the highest identity of 77.22%, with a reported endo-xylanase. The optimum reaction temperature and pH of rSrocXyn10-Ec were pH 7.0 and 60°C, with remarkable stability at 45°C or pHs ranging from 4.5 to 11.0. rBvelXyn11-Ec was most active at pH 6.0 and 50°C, and was stable at 35°C or pH 3.5 to 10.5. Both rSrocXyn10-Ec and rBvelXyn11-Ec showed specific enzyme activities on wheat arabinoxylan (685.83 ± 13.82 and 2809.89 ± 21.26 U/mg, respectively), with no enzyme activity on non-xylan substrates. The Vmax of rSrocXyn10-Ec and rBvelXyn11-Ec were 467.86 U mg-1 and 3067.68 U mg-1, respectively. The determined Km values of rSrocXyn10-Ec and rBvelXyn11-Ec were 3.08 g L-1 and 1.45 g L-1, respectively. The predominant product of the hydrolysis of alkaline extracts from bagasse, corncob, and bamboo by rSrocXyn10-Ec and rBvelXyn11-Ec were xylooligosaccharides. Interestingly, the xylobiose content in hydrolysates by rSrocXyn10-Ec was approximately 80%, which is higher than most reported endo-xylanases. rSrocXyn10-Ec and rBvelXyn11-Ec could be excellent candidates to produce xylooligosaccharides at neutral/near-neutral pHs. rSrocXyn10-Ec also has potential value in the production of xylobiose as a substitute sweetener.

Expression Pattern of Cathelicidins in Dairy Cows During Endometritis and Role of Bovine Endometrial Epithelial Cells in Production of Cathelicidins.

Endometritis is a common bacterial disease of dairy cows. Cathelicidins are host-defense peptides that play important roles in clearance of bacteria. However, the expression pattern of these peptides during endometritis is still unclear. We hypothesize that the levels of bovine cathelicidins increased during endometritis. This study was to investigate the changes of bovine cathelicidins during endometritis. Forty-four post-partum cows (28-35 days after calving) involved in this study were grouped according to the character of vaginal discharge (VD) into three groups. These were (1) cows with clear fluid (n = 8, healthy cows group, N); (2) cows with VD containing <50% off-white mucopurulent material (n = 20, moderate endometritis cows, M); (3) cows with VD containing > 50% yellow or white purulent material (n = 16, severe endometritis cows, S). The blood, VD, and endometrial biopsies samples were collected from each cow to assess the levels of cathelicidin 1-7. Furthermore, bovine endometrial epithelial cells (BEECs) were stimulated with different concentration of Escherichia coli (2 × 106 and 2 × 107 CFU/mL) to detect the cellular source of cathelicidins. Quantitative real-time PCR (RT-qPCR) was used to detect the relative mRNA expression of cathelicidins, and enzyme-linked immune sorbent assay (ELISA) method were used to measure the protein levels. The mRNA and protein levels of cathelicidin 1-7 significantly increased during bovine endometritis (both moderate and severe endometritis), while samples from severe cases showed lower levels of cathelicidins compared to moderate cases. BEECs can express cathelicidin 1-7, and E. coli triggered the release of these proteins. High concentration of E. coli decreased the mRNA and protein levels of cathelicidins. Taken together, our results supported that cathelicidins are released as host defense molecules against the bacteria during bovine endometritis, and BEECs play an active role in expression and production of cathelicidins.

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats.

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 101 copies/µL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.

L'adénocarcinome nasal enzootique est une maladie respiratoire contagieuse chez les chèvres qui est causé par le virus de la tumeur nasale enzootique 2 (ENTV-2). Afin d'augmenter le nombre de méthodes de détection disponibles pour ENTV-2, nous avons développé un test de réaction en chaîne par polymérase en temps réel SYBR Green (SGrPCR) qui cible le gène gag de ENTV-2. La limite basse de détection du test était de 3,68 × 101 copies/µL, cent fois plus sensible que la PCR conventionnelle. La courbe de fusion montrait un seul pic de fusion net à 83 °C, ce qui indiquait qu'il n'y avait pas d'amplification non spécifique ou de formation de dimère d'amorce. Les coefficients de variation intra-essai et inter-essai étaient respectivement de 1,58 % et 1,82 %. Il n'y avait pas de réactivité croisée avec les virus caprins étroitement apparentés (c'est-à-dire le virus orf, le virus de la peste des petits ruminants, le virus de la variole caprine, le virus de la fièvre aphteuse) et les rétrovirus endogènes. En conclusion, le test SGrPCR est spécifique du gène gag de l'ENTV-2 et fournit une approche rapide et sensible pour la détection d'ENTV-2 dans des échantillons cliniques.(Traduit par Docteur Serge Messier).

A rapid and quantitative fluorescent microsphere immunochromatographic strip test for detection of antibodies to porcine reproductive and respiratory syndrome virus.

A fluorescent microsphere-based immunochromatographic strip test (FICT) was developed for the rapid, sensitive, and quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies at the pen-side. The assay was based on the formation of a sandwich immune-complex (anti-pig IgG-PRRSV antibodies-NSP7/N), which was validated by a comparison with IDEXX-ELISA using 3325 clinical specimens. The diagnostic specificity, sensitivity, and accuracy of FICT were 97.28, 93.41, and 94.95%, respectively. FICT showed a good correlation with the virus neutralization assay. Overall, a promising pen-side diagnostic tool was developed for the rapid and quantitative detection of PRRSV antibodies within 15 min.

Decreasing of S100A4 in bovine endometritis in vivo and in vitro.

Endometritis is a prevalent reproductive disease in dairy cows, and is a superficial inflammation of the endometrium. S100 calcium-binding protein A4 (S100A4) is suggested to be implicated in the progression of inflammation. However, to our knowledge, no study has reported the changes of S100A4 during bovine endometritis. The objective of this study was to investigate S100A4 gene expression and protein levels in the uterus with endometritis in dairy cows. Vaginal mucus samples were collected for diagnosis of the severity degree of endometritis and the detection of S100A4 protein content. Blood samples and endometrial biopsies were collected and divided into the control (CN), mild endometrtis (M), and severe endometritis (S) groups according to the characteristics of the vaginal mucus type. The isolated bovine endometrial epithelial cells (BEECs) were challenged with E. coli (2 × 106 CFU/mL, 2 × 107 CFU/mL) or lipopolysaccharide (LPS, 3 and 10 µg/mL) as an inflammatory model. RT-qPCR was used to detect the gene expression levels of S100A4 and cytokines, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α), in tissues or cells. Enzyme-linked immunosorbent assay (ELISA) was used for S100A4 protein level detection in tissues, cells, cell supernatant, vaginal mucus, and serum samples. The results showed that S100A4 gene and protein levels decreased in bovine endometrium with endometritis and in E. coli- or LPS-stimulated BEECs. We failed to detect S100A4 in the cell supernatant, vaginal mucus, and serum samples. This study suggested that S100A4 is a pathogenesis-related protein of endometritis, and decreased expression of S100A4 may pave the way for the development of endometritis in dairy cows.

[Study on clinical efficacy of combination of traditional Chinese medicine and western medicine in treatment of pit viper bites and peripheral blood inflammatory factors].

OBJECTIVE: To observe the clinical efficacy of combination of traditional Chinese medicine and western medicine in the treatment of patient bitten by agkistrodon halys pallas, and the changes in peripheral blood inflammatory factors (hs-PCR, IL-6, TNF-alpha). METHOD: Ninty-eight patients were divided into three groups according to their hospitalization dates: the western medicine group, in which 32 patients were treated with antivenom serum (6 000 U) for five days, once every day; the traditional Chinese medicine group, in which 32 patients were treated with anti pit viper No. 2 concentrated decoction (300 mL), twice to three times every day, for five days; and the combined traditional Chinese medicine and western medicine group, in which 35 patients were treated with the combination of Chinese and Western medicine treatment described above. Then blood samples of all of patients were obtained, and serum factors (hs-PCR, IL-6, TNF-alpha) in peripheral blood were measured by Elisa assay. Another 30 health volunteers were chosen as the normal control group. RESULT: The serum inflammatory factors were significantly higher in all patients of the three groups than that in healthy control before treatment (P < 0.01), and decreased significantly after treatment. In particular, the more remarkable reduction was found in the combined traditional Chinese medicine and western medicine group compared with the western medicine group and the traditional medicine group (P < 0.01). Symptom elimination in the three groups was superior to the western medicine group and the traditional medicine group at the first day and the third day of treatment (P < 0.05, P < 0.01). Total clinical effective rate was 100% in the combined traditional Chinese medicine and western medicine group, 84. 37% in the traditional medicine group and 65.62% in the western medicine group, the clinical effective rate of the combined traditional Chinese medicine and western medicine group was notably superior to that of the western medicine group and the traditional medicine group (P < 0.01). CONCLUSION: The serum inflammatory factors increased significantly in patients bitten by agkistrodon halys pallas. Treatment with the combined traditional Chinese medicine and western medicine can significantly decrease the serum inflammatory factors, and increase clinical effect, with more obvious clinical efficacy compared with the western medicine group and the traditional medicine group.

[Impacts on the appetite regulating factors of infantile anorexia treated with acupuncture at Sifeng (EX-UE 10)].

OBJECTIVE: To explore the dynamic change and clinical efficacy of acupuncture at Sifeng (EX-UE 10) on appetite regulating factors in the serum of infantile anorexia. METHODS: Eighty cases, in compliance with the diagnostic criteria, aged from 3 to 6 years were randomized into an acupuncture group and a medication group, 40 cases in each one. Additionally, a healthy control group (30 cases) was set up. In the acupuncture group, the pricking method was adopted at Sifeng (EX-UE 10) with the three-edged needle. A few light yellow, transparent viscous liquid or blood was squeezed out after pricking. The treatment was given once a week, for 4 weeks totally. In the medication group, erkangning syrup was administered, 3 times a day, for 4 weeks totally. The ghrelin, leptin and neuropeptide Y (NPY), and the clinical efficacy were observed before and after treatment in each group. RESULTS: The levels of ghrelin and NPY before treatment in acupuncture group and the medication group were lower apparently than those in the healthy control group (all P < 0.01), but the level of leptin was higher appa-rently than that in the healthy control group (P < 0.01). After treatment, the levels of ghrelin and NPY were higher apparently than those before treatment in the acupuncture group (both P < 0.01), and the level of leptin was lower apparently than that before treatment (P < 0.01). All of the above indices in the acupuncture group were improved obviously after treatment as compared with those in the medication group (all P < 0.01). The remarkable and effective rate were 82.5% (33/40) and 32.5% (13/40) and the total effective rate were 95.0% (38/40) and 45.0% (18/40) in the acupuncture group and medication group separately, the results in the acupuncture group were superior to the medication group (both P < 0.01). CONCLUSION: Acupuncture at Sifeng (EX-UE 10) effectively promotes the secretion of ghrelin and NPY and inhibit leptin. It effectively promotes appetite for the children and the efficacy is superior to erkangning syrup.