Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Variation in the carbon and oxygen isotope composition of plant biomass and its relationship to water‐use efficiency at the leaf‐ and ecosystem‐scales in a northern Great Plains grassland

Measurements of the carbon (δ¹³C_m) and oxygen (δ¹⁸O_m) isotope composition of C₃ plant tissue provide important insights into controls on water‐use efficiency. We investigated the causes of seasonal and inter‐annual variability in water‐use efficiency in a grassland near Lethbridge, Canada using stable isotope (leaf‐scale) and eddy covariance measurements (ecosystem‐scale). The positive relationship between δ¹³C_m and δ¹⁸O_m values for samples collected during 1998–2001 indicated that variation in stomatal conductance and water stress‐induced changes in the degree of stomatal limitation of net photosynthesis were the major controls on variation in δ¹³C_m and biomass production during this time. By comparison, the lack of a significant relationship between δ¹³C_m and δ¹⁸O_m values during 2002, 2003 and 2006 demonstrated that water stress was not a significant limitation on photosynthesis and biomass production in these years. Water‐use efficiency was higher in 2000 than 1999, consistent with expectations because of greater stomatal limitation of photosynthesis and lower leaf c_i/ca during the drier conditions of 2000. Calculated values of leaf‐scale water‐use efficiency were 2–3 times higher than ecosystem‐scale water‐use efficiency, a difference that was likely due to carbon lost in root respiration and water lost during soil evaporation that was not accounted for by the stable isotope measurements.     

用改进的重叠延伸PCR技术构建三位点突变载体

目的:改进传统重叠延伸PCR方法,实现引入3个不同DNA突变位点的简便的多位点定点突变。方法:根据前期构建的包含人线粒体12S rRNA(NC 01290)3个热点突变位点的野生型质粒序列,利用Muta Primer 2.0软件设计针对3个热点突变位点的3对互补的定点突变引物,以野生型质粒为模板,结合重叠延伸PCR反应和冷冻析出法,产生同时包含3个突变位点的突变目的片段,酶切后克隆到载体中,测序确证是否突变成功。结果:DNA测序证实3个不同突变位点同时成功引入,定点突变载体构建成功。结论:用改进的重叠延伸PCR技术能简便、高效地获得多位点定点突变载体,在分子生物学领域有较高的使用价值。     

Soil microbial and nutrient responses to 7 years of seasonally altered precipitation in a Chihuahuan Desert grassland

Soil microbial communities in Chihuahuan Desert grasslands generally experience highly variable spatiotemporal rainfall patterns. Changes in precipitation regimes can affect belowground ecosystem processes such as decomposition and nutrient cycling by altering soil microbial community structure and function. The objective of this study was to determine if increased seasonal precipitation frequency and magnitude over a 7‐year period would generate a persistent shift in microbial community characteristics and soil nutrient availability. We supplemented natural rainfall with large events (one/winter and three/summer) to simulate increased precipitation based on climate model predictions for this region. We observed a 2‐year delay in microbial responses to supplemental precipitation treatments. In years 3–5, higher microbial biomass, arbuscular mycorrhizae abundance, and soil enzyme C and P acquisition activities were observed in the supplemental water plots even during extended drought periods. In years 5–7, available soil P was consistently lower in the watered plots compared to control plots. Shifts in soil P corresponded to higher fungal abundances, microbial C utilization activity, and soil pH. This study demonstrated that 25% shifts in seasonal rainfall can significantly influence soil microbial and nutrient properties, which in turn may have long‐term effects on nutrient cycling and plant P uptake in this desert grassland.     

Climate change alters ecological strategies of soil bacteria

The timing and magnitude of rainfall events are expected to change in future decades, resulting in longer drought periods and larger rainfall events. Although microbial community composition and function are both sensitive to changes in rainfall, it is unclear whether this is because taxa adopt strategies that maximise fitness under new regimes. We assessed whether bacteria exhibited phylogenetically conserved ecological strategies in response to drying‐rewetting, and whether these strategies were altered by historical exposure to experimentally intensified rainfall patterns. By clustering relative abundance patterns, we identified three discrete ecological strategies and found that tolerance to drying‐rewetting increased with exposure to intensified rainfall patterns. Changes in strategy were primarily due to changes in community composition, but also to strategy shifts within taxa. These moisture regime‐selected ecological strategies may be predictable from disturbance history, and are likely to be linked to traits that influence the functional potential of microbial communities.     

Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. Biotechnol. Bioeng. 2013;110: 2928–2937. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Improved environmental DNA sampling scheme for Alabama sturgeon provides new insight into a species once presumed extinct

Detection of rare species can be challenging and time-consuming using conventional methods, but environmental DNA (eDNA) is becoming a commonly used tool for detection in conservation and management of species. This study demonstrates the utility of the precipitation method (precipitated and preserved in 3 M sodium acetate and 95% ethanol) for collection of eDNA to detect the seasonal distribution of the critically endangered Alabama sturgeon (Scaphirhynchus suttkusi). Surface and benthic water samples were collected across a wider geographic area than previously published for Alabama sturgeon eDNA. Surface and benthic samples both yielded detections and resulted in a similar proportion of positive detections to previous work. However, by sampling a greater portion of the distribution of the Alabama sturgeon, further insight was provided on potential sturgeon movement. The results of the precipitation method show that Alabama sturgeon detections increase during spawning months, and that the fish may be overwintering in the Tombigbee River. High detections from winter benthic samples suggest that habitat choice may play a role in detectability and highlight the need to consider natural history when designing environmental DNA studies. When designing environmental DNA collection for rare species, sampling design should factor in species ecology, habitat use, site characteristics, and specific questions driving the research.     

尿液蛋白富集方法的比较

人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。