Engineering stability into Escherichia coli secreted Fabs leads to increased functional expression

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.     

Bioconjugation of luminescent silicon quantum dots to gadolinium ions for bioimaging applications

Luminescent imaging agents and MRI contrast agents are desirable components in the rational design of multifunctional nanoconstructs for biological imaging applications. Luminescent biocompatible silicon quantum dots (SiQDs) and gadolinium chelates can be applied for fluorescence microscopy and MRI, respectively. Here, we report the first synthesis of a nanocomplex incorporating SiQDs and gadolinium ions (Gd(3+)) for biological applications. The nanoconstruct is composed of a PEGylated micelle, with hydrophobic SiQDs in its core, covalently bound to DOTA-chelated Gd(3+). Dynamic light scattering reveals a radius of 85 nm for these nanoconstructs, which is consistent with the electron microscopy results depicting radii ranging from 25 to 60 nm. Cellular uptake of the probes verified that they maintain their optical properties within the intracellular environment. The magnetic resonance relaxivity of the nanoconstruct was 2.4 mM(-1) s(-1) (in terms of Gd(3+) concentration), calculated to be around 6000 mM(-1) s(-1) per nanoconstruct. These desirable optical and relaxivity properties of the newly developed probe open the door for use of SiQDs in future multimodal applications such as tumour imaging.     

Non-destructive Measurement of Moisture Distribution in Wood during Drying Using Digital X-ray Microscopy

The objective of this study was to develop a nondestructive method by which moisture distribution in wood during drying could be predicted. A newly developed digital X-ray microscope was used to measure the moisture content of wood and its accuracy and resolution was evaluated compared to the classic oven-dry method.

Small green wood specimens of Sugi (Cryptomeria japonica D. Don) were cut and dried under constant temperature and humidity. As the weight was decreasing during drying, X-ray microscope images of cross section were obtained. From these digital images and specimen weight, the moisture content during drying was measured by the two methods. After the shrinkage of the specimen was canceled, the standard error achieved finally was about 1% moisture content within the experimental range. As the image was divided into small subimages, the clear moisture distribution can be seen. It was found that the image divided into 32 × 32 subimages in each size of 0.625 × 0.625 mm might be valid to determine the moisture distribution, and that the drying rate in early wood is larger than in late wood.     

CONTRACTION CHARAClFRlSTlCS OF MOLDED CERAMICS DURING DRYING

ABSTRACT

The characteristics of drvine and contraction (shrinkage) of molded ceramics were expenmentally studied to clarify the effects of drying conditions on contraction behaviors or deformation during drying processes. Mikawa-Shanshu clay molded into spheres or slabs was used as a sample which underwent convective drying by hot air.

Three periods of preheating, constanl rate and falling rate existed, and the contraction was observed mainly in the preheating and constanl rate periods for all samples. It is also observed that the behavior of ihe contraction of molded clay is inflienced significantly by drying conditions. The higher drying rate makes the contraction of clay more remarkable in the preheating and constant rate periods. This behavior could not be explained from the difference of the moisture content distribution among runs. However, it is suggested that the inside parts of molded ceramics may be subjected to the strong compressional stresses and the strain-stress in the sample would Influence the contraction behavior when he gradient of moisture content distribution becomes steeper with increase in the drying rate.     

Facile synthesis of five 2D surface modifiers by highly selective photocyclic aromatization and efficient enhancement of oxygen permselectivities of three polymer membranes by surface modification using a small amount of the 2D surface modifiers

A facile synthesis of novel five 2D (planar) surface modifiers having a triphenylbenzene derivatives as a 2D structure has been achieved by the highly selective photocyclic aromatization reaction. Efficient enhancement of oxygen permselectivities through the three polymer membranes has been achieved by adding a small amount (<5.0 wt%) of the 2D surface modifiers. Among the five 2D surface modifiers, a modifier compound having oligoethylene oxide groups showed the best performance for the enhancement. These improvements were thought to be caused mainly by improvement of the solution selectivity on the membrane surface where the 2D surface modifiers were accumulated. In some of the surface-modified blend membranes, their plots in the PO₂$P_{{\text{O}}_2}$-α graph were over or close to the upper boundary line by Robeson in 1991. Since all the membranes containing the 2D surface modifiers showed better permselectivities than the corresponding substrate membranes, it is very promising for the future.     

Impacts of dam construction on streamflows during drought periods in the Upper Colorado River Basin,Texas

The purpose of this study was to assess the impacts of dam construction on streamflow during a severe drought in the upper Colorado River basin (TX), upstream of Lake Buchanan reservoir. The region has experienced severe, prolonged droughts over the past century. To ameliorate the effects of drought, and increase water storage for use during dry periods, several dams were constructed on the mainstem and tributaries of the Colorado River upstream of Lake Buchanan since the late‐1940s. Analysis of flow at the gauge above Lake Buchanan indicated streamflow was significantly reduced during the recent drought (2009–2014), compared with streamflows during the ‘drought of record’ (1950–1957). The construction of these upstream dams reduced streamflow by intercepting and storing water. It is concluded that building reservoirs in the western portion of the basin, largely in response to past droughts, although increasing water supplies for local uses, exacerbated the downstream effects of the hydrological drought, essentially making it the worst in recorded history.     

高效液相色谱-串联质谱法测定烟草中马来酰肼残留量

建立了高效液相色谱-串联质谱（LC-MS/MS）测定烟草中马来酰肼农药残留的方法。烟草样品经盐酸溶液加热回流萃取,冷却后过膜稀释,多反应监测正离子模式测定,氘代马来酰肼同位素内标定量。结果表明,马来酰肼的定量限为0.27 mg/kg,加标回收率在90.3%~101.5%之间,相对标准偏差（RSD）小于5.5%;采用该方法对12个烟草样品进行检测,马来酰肼在其中10个样品中无检出,将有检出的2个样品与标准方法的测定值进行比较,结果显示两组数据的一致性较好,无显著性差异。该方法前处理简单、灵敏度高、稳定性好,适用于烟草样品中马来酰肼残留量的测定。     

三维数值模拟技术在深部大型发电站厂房信息化施工中的应用

提出一种可以综合考虑围岩节理分布特性、爆破施工损伤以及施工过程的新三维快速数值仿真模拟技术,并将其成功地应用于日本宫崎县120×10~4kW蓄能水力发电工程大型地下厂房的信息化施工中。依据多种测试手段,对开挖后围岩的稳定性和变形进行实时监测,并对数值模型进行三维反演分析和修正,比较准确地预测了围岩的动态变化过程,并对支护结构设计的可靠性和合理性进行了详细的论证。     

A preliminary study on the occurrence and behavior of sulfonamides, ofloxacin and chloramphenicol antimicrobials in wastewaters of two sewage treatment plants in Guangzhou, China

Wastewater samples collected from two sewage treatment plants (STPs) in Guangzhou, China were acidified, solid-phase extracted (SPE) with Oasis HLB cartridges, followed by instrumental measurement by high performance liquid chromatography (HPLC) coupled with a diode array UV detector (DAD) and a fluorescence detector (FLD) for the occurrence and fate of antimicrobial compounds sulfadiazine (SDZ), sulfamethoxazole (SMX), ofloxacin (OFX) and chloramphenicol (CAP). Antimicrobials have been detected at 5.10-5.15, 5.45-7.91, 3.52-5.56 and 1.73-2.43 microg L(-1) for SDZ, SMX, OFX and CAP in the raw sewages of the two STPs, respectively. The concentrations of antimicrobials do not show substantial changes after preliminary mechanical sedimentation. No quantifiable sulfonamides and chloramphenicol have been identified, and >85% of ofloxacin has been removed in the effluents after activated sludge treatment in the two STPs, indicating that activated sludge treatment is effective and necessary to remove antimicrobial substances in municipal sewage.     

Biocomposites composed of epoxidized soybean oil cured with terpene‐based acid anhydride and cellulose fibers

Epoxidized soybean oil (ESO) was cured with a terpene‐based acid anhydride (TPAn) at 150°C, and the thermal and mechanical properties of the cured product were compared with ESO cured with hexahydrophthalic anhydride (HPAn), maleinated linseed oil (LOAn), or thermally latent cationic polymerization catalyst (CPI). The ESO‐TPAn showed a higher glass transition temperature (67.2°C) measured by dynamic mechanical analysis than ESO‐HPAn (59.0°C), ESO‐LOAn (?41.0°C), and ESO‐CPI (10.0°C). The storage modulus at 20°C of ESO‐TPAn was higher than those of ESO‐LOAn and ESO‐CPI. Also, ESO‐TPAn showed higher tensile strength and modulus than the other cured ESOs. Regarding the biodegradability measured by biochemical oxygen demand in an activated sludge, ESO‐TPAn possessed some biodegradability, which was lower than that of ESO‐LOAn. Next, biocomposites composed of ESO‐TPAn and regenerated cellulose (lyocell) fabric were prepared by compression molding method. The tensile strength of ESO‐TPAn/lyocell composites increased with increasing fiber content. The tensile strength and modulus of ESO‐TPAn/lyocell composite with fiber content 75 wt % were 65 MPa and 2.3 GPa, which were three times higher than those of ESO‐TPAn. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008