Laboratory Evolution of GH11 Endoxylanase Through DNA Shuffling: Effects of Distal Residue Substitution on Catalytic Activity and Active Site Architecture.

Endoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand. The mutant library of GH11 endoxylanase was constructed via DNA shuffling by using the catalytic domain of Bacillus amyloliquefaciens xylanase A (BaxA) and Thermomonospora fusca TF xylanase A (TfxA) as parents. A total of 2,250 colonies were collected and 756 of them were sequenced. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. The specific activity of DS153, DS241, and DS428 in the optimal condition was 4.54, 4.35, and 3.9 times compared with the recombinant BaxA (reBaxA), respectively. The optimum temperature of the three mutants was 50°C. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. The catalytic efficiency of DS153, DS241, and DS428 enhanced as well, while their sensitivity to recombinant rice xylanase inhibitor (RIXI) was lower than that of reBaxA. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose-xylopentaose from oat spelt, birchwood, and beechwood xylan. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase activity. The tertiary structure of BaxA was not altered under the substitution of distal residues (N29S, S31R, and I51V); it induced slightly changes in active site architecture. The distal impact rescued the BaxA from native conformation ("closed state") through weakening interactions between "gate" residues (R112, N35 in DS241 and DS428; W9, P116 in DS153) and active site residues (E78, E172, Y69, and Y80), favoring conformations with an "open state" and providing improved activity. The current findings would provide a better and more in-depth understanding of how distal single residue substitution improved the catalytic activity of xylanase at the atomic level.

Effect of CYP2C19 Gene Polymorphisms on Proton Pump Inhibitor, Amoxicillin, and Levofloxacin Triple Therapy for Eradication of Helicobacter Pylori.

BACKGROUND The effects of PPI are variable owing to the CYP2C19 polymorphisms. However, whether the polymorphisms could affect the Hp eradication efficacy of triple therapy is still not clear. The present study aimed to assess the effects of CYP2C19 gene polymorphisms on proton pump inhibitor (PPI), amoxicillin, and levofloxacin triple therapy for Helicobacter pylori (Hp) eradication. MATERIAL AND METHODS We randomly assigned 160 Hp-positive patients with chronic gastritis to 2 groups to receive either 20 mg bid omeprazole (OAL group, n=80) or 10 mg bid rabeprazole (RAL group, n=80), combined with 1000 mg bid amoxicillin and 500 mg qd levofloxacin. The 2 groups were treated for 10 days. The CYP2C19 genotypes included wild-type, M1 mutant gene (*2, the mutation of exon 5), and M2 mutant gene (*3, the mutation of exon 4) identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFIP). According to CYP2C19 genotype combinations, the patients were divided into extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) subgroups. The eradication efficacy of Hp was evaluated by 14C-UBT at 28 days after treatment. RESULTS The trial was completed by 155 patients. Hp eradication rates in OAL and RAL groups were 78.2% and 88.3%, respectively, on per-protocol (PP) analysis, indicating no significant difference (P>0.05). Regarding CYP2C19 genotypes, eradication rates of 60.7%, 84.2%, and 100% were obtained for EM, IM, and PM subgroups, respectively, of the OAL group. EM group eradication rates were significantly lower than IM and PM group values (P<0.05). In the RAL group, no such difference was observed (P>0.05). Hp eradication rates were significantly lower in the EM subgroup of the OAL group compared with that of the RAL group. CONCLUSIONS Hp eradication rates were higher in the RAL group than in OAL-treated patients. Interestingly, omeprazole-based therapy was significantly affected by the CYP2C19 genotype, unlike the rabeprazole-based therapy.

[Vertical distribution and relationship between 210Pb(ex) activities and nutrients in sediment cores of two different eutrophication level lakes].

The 210Pb(ex) activities and nutrient (TOC, TN and TP) contents in sediment cores of Fuxian Lake and Dianchi Lake were measured by traditional methods, as well as their vertical distribution and relationship were also comparatively analyzed in this paper. The study results indicated that the vertical distributions of 210Pb(ex) and nutrients were significantly different between Fuxian Lake and Dianchi Lake. The variation amplitude of 210Pb(ex) activities in Dianchi Lake was higher than that in Fuxian Lake. The disordered distribution characteristics of 210Pb(ex) in Dianchi Lake surface sediments were closely related to physicochemical migration of lead caused by human activities. The variation trends of nutrients in sediment cores were corresponded to local natural evolution and human activities in different historical periods. Relationship between 210Pb(ex) activities and nutrients were mainly affected by the nutrition level of lakes. The greater the lake eutrophication level was, the stronger the correlation was found. To the individual nutrient indicators, similar characteristics were shown in the two lakes, and the order was TOC > TP > TN.

[Photosynthetic functions and chlorophyll fast fluorescence characteristics of five Pinus species].

A comparative study was made on the needle morphological characteristics, photosynthetic rate, and chlorophyll fast fluorescence induction curves of five representative Pinus species P. parvifiora, P. armandii, P. bungeana, P. tabuliformis, and P. densiflora. Significant differences were observed in the needle morphological characteristics among the five species. P. tabuliformis had the longest needle length and highest needle density, whereas P. bungeana had the highest chlorophyll content. P. densiflora and P. parvifiora had the maximum and minimum photosynthetic rate, respectively. There was a positive correlation between the photosynthetic rate and stomatal conductance across the five species. The differences in the chlorophyll fast fluorescence induction curves of the five species were mainly manifested in J-step and I-step. Although the five species had similar values of Fv/Fm, Fv/Fo and Tfm, P. parviflora had significantly higher values of dV/dt(o), dVG/d(o), V and Vi, but lower energy flux ratio psi(o), phiEo and phiRo, compared with the other four species. The low PSII activity and efficiency of P. parviflora might relate to its smallest Sm, Sm/Tfm and N. P. densiflora and P. parvifiora had the maximum and minimum vitality indices PI(ABS/CSo/CSm) and DF, respectively, and there existed significant positive correlations between the PI(CSo) and PI(CSm) and the net photosynthetic rate of the five species, suggesting that PI(CSo) and PI(CSm) could be used to estimate the photosynthetic activity of Pinus trees.

[Estimation models of vegetation fractional coverage (VFC) based on remote sensing image at different radiometric correction levels].

The images of post atmospheric correction reflectance (PAC), top of atmosphere reflectance (TOA), and digital number (DN) of a SPOT5 HRG remote sensing image of Nanjing, China were used to derive four vegetation indices (VIs), i. e., normalized difference vegetation index (NDVI), transformed vegetation index (TVI), soil-adjusted vegetation index (SAVI), and modified soil-adjusted vegetation index (MSAVI), and 36 VI-VFC relationship models were established based on these VIs and the VFC data obtained from ground measurement. The results showed that among the models established, the cubic polynomial models based on NDVI and TVI from PAC were the best, followed by those based on SAVI and MSAVI from DN, with the accuracy being slightly higher than that of the former two models when VFC > 0.8. The accuracy of these four models was higher in middle-densely vegetated areas (VFC = 0.4-0.8) than in sparsely vegetated areas (VFC = 0-0.4). All the established models could be used in other places via the introduction of calibration models. In VI-VFC modeling, using VIs derived from different radiometric correction levels of remote sensing image could help mining valuable information from remote sensing image, and thus, improving the accuracy of VFC estimation.