β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

Assessing the conservation effectiveness of wetland protected areas in Northeast China

The evaluation of conservation effectiveness for wetland protected areas (WPAs) is essential to underpin knowledge-based conservation policies and funding decisions by government and managers. In this paper, the conservation effectiveness for 28 WPAs in Northeast China from 2000 to 2012 was quantitatively evaluated using landsat thematic mapper image data and a maximum entropy model (Maxent). The spatial distribution of conservation effectiveness and the influence of human activities on conservation effectiveness were determined by combining a landscape development intensity (LDI) index and spatial analysis in a geographical information system. The results showed that the natural wetland area of all WPAs in Northeast China declined by 11.5 % and the conservation effectiveness of most of these WPAs decreased between 2000 and 2012. A significant negative correlation between the LDI index and conservation effectiveness (r = ?0.824, p < 0.01) suggested that human activities were responsible for the low conservation effectiveness of WPAs. The WPAs with the high conservation effectiveness were mainly located in the Da-Xing’an and Xiao-Xing’an Mountains, where anthropogenic activities were limited. The reduction in the conservation effectiveness of WPAs in the Songnen and Sanjiang Plains, which showed the most degradation, was due to conversion of wetlands to croplands. This research offers an efficient and effective method to evaluate the conservation effectiveness of WPAs. The results of this study will inform future ecological conservation and management of WPAs in China.     

Expression,purification and characterization of a vascular endothelial growth factor fusion protein

Objectives

To prepare recombinant tPep-(vascular endothelial growth factor) VEGF-B and assess its biological activity.

Results

This new VEGF fusion protein was constructed using a targeting peptide and prepared using E.coli. The tPep-VEGF-B was refolded from inclusion bodies and purified using affinity chromatography. Its bioactivity was determined in vitro using proliferation assay and wounding healing assay, and in vivo in zebrafish. By using the optimized downstream process, recombinant tPep-VEGF-B can be obtained with a purity of >90 % and a yield of 80 mg protein/l culture medium. The refolded protein is highly effective in promoting cell migration in vitro and in enhancing angiogenesis in vivo.

Conclusion

We have constructed a new VEGF fusion protein with potential therapeutic application in treating metabolic diseases.

     

Optimal secretion of alkali-tolerant xylanase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by signal peptide screening

Xylanases are industrially important enzymes for xylan digestion. We experimentally screened over 114 Sec and 24 Tat pathway signal peptides, with two different promoters, for optimal production of an alkaline active xylanase (XynBYG) from Bacillus pumilus BYG in a Bacillus subtilis host. Though both promoters yielded highly consistent secretion levels (0.97 Pearson correlation coefficient), the Sec pathway was found to be more efficient than the Tat pathway for XynBYG secretion. Furthermore, the optimal signal peptide (phoB) for XynBYG secretion was found to be different from the optimal peptides for cutinase and esterase reported in previous studies. A partial least squares regression analysis further identified several statistically important variables: helical properties, amino acid composition bias, and the discrimination score in Signal P. These variables explain the observed 23 % variance in the secretion yield of XynBYG by the different signal peptides. The results also suggest that the helical propensity of a signal peptide plays a significant role in the beta-rich xylanase, but not in the helix-rich cutinase, suggesting a coupling of the conformations between the signal peptide and its cargo protein for optimal secretion.     

Short-finned pilot whales in the South China Sea: Insights into regional distribution,movement pattern,and habitat characteristics

Little scientific knowledge is available for short-finned pilot whales in the South China Sea (SCS). Here, we integrated historical records, ship-based observation, and satellite-tagging data to investigate ecological and behavioral characteristics of pilot whales in the SCS. Historical records since the 1990s showed the widespread spatial occurrence of pilot whales along the coastal SCS, suggesting that the species may inhabit this region. During our dedicated ship-based surveys (2019–2022) in the SCS, 19 groups of pilot whales were encountered in Xisha-Zhongsha waters, which supported the hypothesis inferred from historical data. We believe that Xisha-Zhongsha waters are important habitats for pilot whales, demonstrated by the large group size (M = 44.7 ± 42.4 SD individuals), the high proportion of recorded maternal groups (17/19), mixed-species associations, and deep-water habitat characteristics. During field surveys, we conducted satellite-tagging experiments on two adult pilot whales, with tracking periods of 35 and 13 days, respectively. Satellite-tracking data provided evidence of possible residency of the tagged whales, as both displayed overlapping ranges and nondirectional movement. This research provides preliminary baseline data on occurrence, distribution, movement, habitat use, and likely residency of pilot whales in the SCS, which can facilitate future research and conservation effort.     

城乡交错带土壤氮素空间分布及其影响因素

城乡交错带土壤氮素是城乡生态系统中最重要的氮源与氮汇,但是城市化下的土壤氮素分布及其影响机制还不清楚,基于3S平台研究了土壤氮素在成都西郊城乡交错带的空间分布特征及城市化对土壤氮素的影响。结果表明,研究区内土壤全氮(STN)、硝态氮(NO_3~--N)和铵态氮(NH_4~+-N)含量均值分别为(1.46±0.06)g/kg、(50.04±3.59)mg/kg和(6.72±0.53)mg/kg。区内土壤氮素含量从近郊向远郊逐渐增高,STN和NO_3~--N含量为中部高于南北部,NH_4~+-N含量则由西北部和东南部向中部递增。方差分析表明,区内不同土地利用方式下STN、NO_3~--N和NH_4~+-N含量差异显著(P0.05)。回归分析显示STN含量与建筑密度(BD)、道路密度(RD)均呈现显著线性负相关(P0.05),NO_3~--N含量与道路密度呈极显著线性负相关(P=0.001),与建筑密度关系不明显(P=0.217)。土壤NH_4~+-N与建筑密度呈显著负线性相关(P=0.001),与道路密度呈显著指数相关关系(P=0.021)。研究结果显示城市发展使得城乡交错带土壤氮素含量降低,这种影响伴随着建筑面积的增加,道路长度的增加而加强。     

Depletion of Acidic Phosphopeptides by SAX To Improve the Coverage for the Detection of Basophilic Kinase Substrates

The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.     

寡雄腐霉发酵液的动物安全性及其对黄瓜的防病促生效应

【目的】生物农药安全无害,环境友好。为了评价寡雄腐霉发酵液(Pythium oligandrum broth,POB)的动物安全性和防病促生效应,研制高效、无害的生物农药。【方法】试验利用自主分离的寡雄腐霉生防菌株(P.oligandrum CQ2010)制备POB,通过动物试验、拮抗试验、盆栽和生产试验,研究了POB的动物毒性,对黄瓜蔓枯病的防治作用,以及对黄瓜生长、产量和品质的影响。【结果】用大剂量的POB灌胃给药对小鼠体重增长无显著影响,其外观和行为均无异常,组织器官也未见病理改变。POB对甜瓜球腔菌的抑制率为51.95%,药效介于1∶800的百菌清溶液和1∶200的甲基托布津溶液之间。在黄瓜幼苗接种甜瓜球腔菌前后喷施POB,叶片丙二醛含量下降,过氧化物酶和超氧化物歧化酶活性增强,发病率和病情指数均显著降低,相对防治效果达到54.8%–64.1%,故POB能减轻病原菌对细胞膜的伤害,激发防御性生理反应,增强黄瓜植株的抗病能力。此外,POB处理提高叶绿素含量,增强根系活力,增加植株氮、磷、钾吸收量,促进黄瓜植株生长,生物量和果实产量分别提高81.10%和11.58%。POB还使黄瓜果实Vc和可溶性糖提高,硝酸盐含量降低。【结论】寡雄腐霉发酵液对动物安全无毒,能有效防治黄瓜蔓枯病,促进黄瓜生长,提高产量品质。     

A Duplex Real-Time PCR Assay for the Simultaneous Detection of Porcine Circovirus 2 and Circovirus 3

Porcine circoviruses (PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry.     

Enzymatic synthesis and antitumor evaluation of mono- and diesters of 3-O-β-D-galactopyranosyl-sn-glycerol

Lipase-catalyzed synthesis of mono- and diesters of 3-O-β-D-galactopyranosyl-sn- glycerol (β-GG) with caproic acid was performed in acetone. The simultaneous production of 1(6’)-monoesters and 1,6’-diesters of β-GG was achieved in this reaction. In order to improve the yield of β-GG esters, four process parameters, enzyme concentration (15?～?25?mg/mL), and substrate molar ratio (caproic acid: β-GG=?1.60?～?2.00?mmol: 0.10?mmol), reaction temperature (40?～?60?°C), and reaction time (8?～?12?h), were optimized via response surface methodology (RSM) employing a three-level-four-variable central composite design. Results showed that enzyme concentration had the most significant (p?β-GG esters. The optimal reaction conditions in acetone were given as follows: Novozyme435 concentration 18.65?mg/mL, molar rate of caproic acid to β-GG 19.46:1, reaction temperature 48?°C, and reaction time 9.83?h. The yield of β-GG esters reached 88.08% under above optimized conditions, which was very close to the predicted value 87.95%. The molar ratio of monoester to diesters was 0.39:0.61. β-GG esters with other fatty acyl chains were synthesized based on the optimized conditions. In vitro antitumor activity indicated that the antitumor activity of β-GG esters was dependent on the nature of fatty acids, such as the length of acyl chain, the degree of saturation, as well as the number of acyl chain.