Improving the positional accuracy of the goniometer on the Philips CM series TEM

We have developed a method to improve the accuracy for absolute relocation of a target specimen using the goniometer on a Philips transmission electron microscope. We have achieved this by characterizing the performance of the Philips compustage, modeling its behavior, and using this model to calculate the goniometer movements required for accurate target relocation. This resulted in a 10-fold improvement in the positioning accuracy of the goniometer.     

The general form of 0-1 programming problem based on DNA computing

DNA computing is a novel method of solving a class of intractable computational problems, in which the computing speeds up exponentially with the problem size. Up to now, many accomplishments have been made to improve its performance and increase its reliability. In this paper, we solved the general form of 0-1 programming problem with fluorescence labeling techniques based on surface chemistry by attempting to apply DNA computing to a programming problem. Our method has some significant advantages such as simple encoding, low cost, and short operating time.     

Parallel cascade identification and its application to protein family prediction

Parallel cascade identification is a method for modeling dynamic systems with possibly high order nonlinearities and lengthy memory, given only input/output data for the system gathered in an experiment. While the method was originally proposed for nonlinear system identification, two recent papers have illustrated its utility for protein family prediction. One strength of this approach is the capability of training effective parallel cascade classifiers from very little training data. Indeed, when the amount of training exemplars is limited, and when distinctions between a small number of categories suffice, parallel cascade identification can outperform some state-of-the-art techniques. Moreover, the unusual approach taken by this method enables it to be effectively combined with other techniques to significantly improve accuracy. In this paper, parallel cascade identification is first reviewed, and its use in a variety of different fields is surveyed. Then protein family prediction via this method is considered in detail, and some particularly useful applications are pointed out.     

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.     

Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues

Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions.     

A computational method for NMR-constrained protein threading.

Protein threading provides an effective method for fold recognition and backbone structure prediction. But its application is currently limited due to its level of prediction accuracy and scope of applicability. One way to significantly improve its usefulness is through the incorporation of underconstrained (or partial) NMR data. It is well known that the NMR method for protein structure determination applies only to small proteins and that its effectiveness decreases rapidly as the protein mass increases beyond about 30 kD. We present, in this paper, a computational framework for applying underconstrained NMR data (that alone are insufficient for structure determination) as constraints in protein threading and also in all-atom model construction. In this study, we consider both secondary structure assignments from chemical shifts and NOE distance restraints. Our results have shown that both secondary structure assignments and a small number of long-range NOEs can significantly improve the threading quality in both fold recognition and threading-alignment accuracy, and can possibly extend threading's scope of applicability from homologs to analogs. An accurate backbone structure generated by NMR-constrained threading can then provide a great amount of structural information, equivalent to that provided by many NMR data; and hence can help reduce the number of NMR data typically required for an accurate structure determination. This new technique can potentially accelerate current NMR structure determination processes and possibly expand NMR's capability to larger proteins.     

Comparative analysis of gene expression level by quantitative real-time PCR has limited application in objects with different morphology

qRT-PCR is a generally acknowledged method for gene expression analysis due to its precision and reproducibility. However, it is well known that the accuracy of qRT-PCR data varies greatly depending on the experimental design and data analysis. Recently, a set of guidelines has been proposed that aims to improve the reliability of qRT-PCR. However, there are additional factors that have not been taken into consideration in these guidelines that can seriously affect the data obtained using this method. In this study, we report the influence that object morphology can have on qRT-PCR data. We have used a number of Arabidopsis thaliana mutants with altered floral morphology as models for this study. These mutants have been well characterised (including in terms of gene expression levels and patterns) by other techniques. This allows us to compare the results from the qRT-PCR with the results inferred from other methods. We demonstrate that the comparison of gene expression levels in objects that differ greatly in their morphology can lead to erroneous results.     

An improved surface-based method for DNA computation

DNA computing is a novel method for solving a class of intractable computational problems, in which the computing time can grow exponentially with problem size. Up to now, many accomplishments have been achieved to improve its performance and increase its reliability, among which a surface-based method is an efficient candidate. In this paper, the surface-based approach proposed by Liu, Q., Wang, L., Frutos, A.G., Condon, A.E., Corn, R.M., and Smith, L.M., 2000, DNA computing on surfaces. Nature 403, 175-179 is analyzed and an improved surface-based method for DNA computation (i.e. the hybrid DNA/optical computing method) is proposed. Compared with Liu et al.'s approach, our method has some significant advantages such as low cost, short operating time, reusable surface and simple experimental steps. Moreover, the concept of combining easily patterned DNA computing steps with equally parallel, but generally uniform and not easily patterned optical computing steps is an important new direction.     

Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation

Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products.     

Simple screening method for improving membrane protein thermostability

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.     

A single cell approach to problems of cell lineage and commitment during embryogenesis of Drosophila melanogaster

The mechanisms leading to the commitment of a cell to a particular fate or to restrictions in its developmental potencies represent a problem of central importance in developmental biology. Both at the genetic and at the molecular level, studies addressing this topic using the fruitfly Drosophila melanogaster have advanced substantially, whereas, at the cellular level, experimental techniques have been most successfully applied to organisms composed of relatively large and accessible cells. The combined application of the different approaches to one system should improve our understanding of the process of commitment as a whole. Recently, a method has been devised to study cell lineage in Drosophila embryos at the single cell level. This method has been used to analyse the lineages, as well as the state of commitment of single cell progenitors from various ectodermal, mesodermal and endodermal anlagen and of the pole cells. The results obtained from a clonal analysis of wild-type larval structures are discussed in this review.     

Measuring low levels of protein aggregation by sedimentation velocity

The required performance of an analytical method depends on the purpose for which it will be used. As a methodology matures, it may find new application, and the performance demands placed on the method can increase. Sedimentation velocity analytical ultracentrifugation (SV-AUC) has a long and distinguished history with important contributions to molecular biology. Now the technique is transitioning into industrial settings, and among them, SV-AUC is now used to quantify the amount of protein aggregation in biopharmaceutical protein products, often at levels less than 1% of the total protein mass. In this paper, we review recent advances to SV methodology which have been shown to improve quantitation of protein aggregation. Then we discuss the performance of the SV method in its current state, with emphasis on the precision and quantitation limit of the method, in the context of existing industrial guidance on analytical method performance targets for quantitative methods.     

基于分子信标的DNA计算

DNA计算是解决一类难以计算问题的一种新方法,这种计算随着问题的增大可以呈指数增长．迄今为止,许多研究成果已经成功地提高了它的性能和增加了它的可行性,本文在基于表面的DNA计算中采用了分子信标编码策略,并对分子信标在与对应的补链杂交形成双链时的受力进行分析,给出3-SAT问题的另一种解法．这种方法比现有的方法更有效,更具发展前景．因为它具有编码简单;耗材底;操作时间短;技术先进等优点．本文尝试了分子生物学,光学和力学的结合．这一工作为DNA计算能解决NP一完全问题提供了更有力的依据．     

基于神经网络的复介电常数重建算法

提出将一种神经网络的方法应用于微波断层成象,重建复的介电常数图象。微波断层成象发展了二十多年,仍然处于实验阶段,这是因为必须求解逆散射问题,它具有非适应性和非线性。为了改善其非适应性及非线性,引入适当的正则化过程并且应用神经网络的方法来求解这个问题。数值模拟实例证明了此方法的有效性。     

环介导等温扩增技术改进的研究进展

环介导等温扩增法(LAMP)是近年发展起来的新型核酸检测技术,因其检测特异性好、灵敏度高、时间短、无需热循环设备而在病原体检测中得到广泛应用。基于该技术进行的改造和改良也层出不穷,本文从该技术的方法改造、缩短反应时间和简化模板预处理、多重扩增产物分析、防止假阳性污染四个方面对近几年LAMP技术的发展进行综述,为今后以该技术平台为基础的创新和应用提供理论依据。     

昆虫神经元染色的银增感法

NiCl2逆行染色是观察昆虫神经元结构的一个重要方法。这一方法不足之处是使一些神经末梢的分支不清晰,但可以通过神经元染色后的银增感方法来弥补。作者借对双斑蟋蟀Gryllus bimaculatusde Geet产卵瓣内的感觉神经元染色后的银增感介绍了这一方法,包括材料的解剖、染色、固定、增感、脱水、透明等。     

Intelligent Scheduling for Underground Mobile Mining Equipment

Many studies have been carried out and many commercial software applications have been developed to improve the performances of surface mining operations, especially for the loader-trucks cycle of surface mining. However, there have been quite few studies aiming to improve the mining process of underground mines. In underground mines, mobile mining equipment is mostly scheduled instinctively, without theoretical support for these decisions. Furthermore, in case of unexpected events, it is hard for miners to rapidly find solutions to reschedule and to adapt the changes. This investigation first introduces the motivation, the technical background, and then the objective of the study. A decision support instrument (i.e. schedule optimizer for mobile mining equipment) is proposed and described to address this issue. The method and related algorithms which are used in this instrument are presented and discussed. The proposed method was tested by using a real case of Kittilä mine located in Finland. The result suggests that the proposed method can considerably improve the working efficiency and reduce the working time of the underground mine.     

MMP1 gene expression enhances myoblast migration and engraftment following implanting into mdx/SCID mice

Myoblast transplantation (MT) is a method to introduce healthy genes into abnormal skeletal muscle. It has been considered as a therapeutic modality in the last few decades for diseases such as Duchenne Muscular Dystrophy (DMD). However, challenges including cell death and poor graft engraftment have limited its application. The current experiment utilizes MMP1 gene transfer to improve the efficacy of myoblast transplantation into the diseased dystrophic skeletal muscle of mdx mice. Our results indicated that MMP1 expression can promote myogenic differentiation and fusion capacities, increase migration of MMP1 expressing myoblasts in vitro, as well as improve engraftment of dystrophin positive myofibers in vivo. Taken together, our observation suggests that the addition of MMP1 can overcome limitations in MT and improve its clinical efficacy.     

A simplified hydroethidine method for fast and accurate detection of superoxide production in isolated mitochondria

Because superoxide is involved in various physiological processes, many efforts have been made to improve its accurate quantification. We optimized and validated a superoxide-specific and -sensitive detection method. The protocol is based on fluorescence detection of the superoxide-specific hydroethidine (HE) oxidation product, 2-hydroxyethidium. We established a method for the quantification of superoxide production in isolated mitochondria without the need for acetone extraction and purification chromatography as described in previous studies.