Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot

To increase the throughput of protein identification and characterization in proteome studies, we investigated three methods of performing protein digestion in parallel. The first, which we term "one-step digestion-transfer" (OSDT), is based on protein digestion during the transblotting process. It involves the use of membranes containing immobilized trypsin which are intercalated between the gel and a PVDF collecting membrane. During electrotransfer, some digestion of the transferred proteins occurs, although poorly for basic and/or high molecular weight proteins. The second method is based on "in-gel" digestion of all proteins in parallel and termed "parallel in-gel digestion" (PIGD) to denote this fact. The PIGD led to more efficient digestion of basic and high molecular weight proteins (> 40,000) but suffered from a major drawback: loss of resolution for low molecular weight polypeptides (< 60,000) through diffusion during the digestion process. The third method examined was the combination of PIGD and OSDT procedures. This combination, called "double parallel digestion" (DPD), led to greatly improved digestion of high molecular weight and basic proteins without losses of low molecular weight polypeptides. Peptides liberated during transblotting of proteins through the immobilized trypsin membrane were trapped on a PVDF membrane and identified by mass spectrometry in scanning mode.     

Pattern-driven green adaptation of process-based applications and their runtime infrastructure

Business processes are a key aspect of modern organization. In recent years, business process management and optimization has been applied to different cross-cutting concerns such as security, compliance, or Green IT, for example. Based on the ecological characteristics of a business process, proper environmentally sustainable adaptation strategies can be chosen to improve the total environmental impact of the business process. We use ecological sustainable adaptation strategies that are described as green business process patterns. The application of such a green business process pattern, however, affects the business process layer, the application component and the infrastructure layer. This implies that changes in the application infrastructure also need to be considered. Hence, we use best practices of cloud application architectures which are described as Cloud patterns. To guide developers through the adaptation process we propose a pattern-based approach in this work. We correlate Cloud patterns relevant for sustainable business processes to green business process patterns and organize them within a classification. To provide concrete implementation support we further annotate these Cloud patterns to application component models that are described with the topology and orchestration specification for cloud applications (TOSCA). Using these annotations, we describe a method that provides the means to optimize business processes based on green business process patterns through adapting the implementation of application components with concrete TOSCA implementation models.     

Method for the pre-dimensioning of beveloid gears

Conical involute gears – also referred to as beveloid gears – are based on cylindrical involute gears, as both gear geometries are based on a basic rack. They differ from cylindrical gears in that the beveloid gears’ addendum modification along the face width is not constant, but linearly variable. This allows beveloid gears to transfer mechanical power with parallel, intersecting or even skewed axes up to shaft angles of approximately 20°, whereas bevel or hypoid gears are applied with larger shaft angles.Compared with both cylindrical and bevel gears, there is less knowledge available concerning the design of beveloid gears. Existing tools for contact analysis are based on predetermined main gearing data. Therefore a method to pre-dimension main beveloid gearing data based on application requirements was developed. Initially, equivalent cylindrical gears are dimensioned and then transformed into beveloid gears with the required mounting positions. The method involves pre-optimizing of beveloid gears with several targets. Firstly, to comply with selected boundaries of the addendum modification coefficient, as well as to minimize ease-off between the tooth flanks achieved by a centric contact pattern and ensure adequate tip clearance by reducing tip circles.The method was implemented in a software environment for tooth contact analysis and applied to a wide range of data sets. The evaluation demonstrated the efficient design of the main data of beveloid gears including pre-optimization to achieve the objectives mentioned previously. In further optimizations, the microgeometry of beveloid gears must be adapted regarding the gap that occurs between the flanks.     

烧结熔剂高温性能的研究

为了解熔剂高温分解特性和高温反应特性在烧结工艺中的重要作用,通过熔剂高温分解特性试验,得到白云石和石灰石的分解温度,发现白云石失重率较高,产生气孔较多,有利于提高烧结料层透气性。烧结前对白云石和石灰石进行轻烧水化,有利于提高烧结矿质量;通过熔剂高温反应特性试验,发现石灰石和生石灰的变形温度和半球温度相差不大,白云石的变形温度和半球温度较高,且温度区间较大,石灰石和生石灰的流动性优于白云石。     

无油螺杆压缩机装配间隙的优化设计

基于无油螺杆压缩机的结构形式,利用有限元分析方法,建立三维模型,对转子的热、力变形进行分析.采用流固耦合的分析方法,优化螺杆转子的温度场分布.分析结果表明,螺杆转子的温度场分布更接近实际,同时压缩机冷态间隙的设置不仅需要考虑转子的热膨胀,还需要考虑气缸体的热力变形.在工程实际中,根据有限元分析的结果,对压缩机各泄漏间隙的冷、热态装配值进行对比,调整实际间隙值,从而实现间隙的优化设计.     

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.     

Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage

Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

Evaluation of possible interference of anti-inflammatory drugs upon scintigraphic imaging with macrophage targeting 99mTc-J001X: effects of methylprednisolone, dexamethasone, indomethacin and methotrexate

J001X, an acylated poly-(1,3)-galactoside isolated from Klebsiella pneumoniae proteoglycan, has been developed to target cells from the monocyte-macrophage lineage. Recent experimental work and initial clinical trials have proved the potential of this molecule labeled with 99mTc for the scintigraphy of inflammatory foci. In a model of radiation-induced inflammation in pigs, the scintigraphic contrast was observed to be very sensitive to a single injection of methylprednisolone given 12 h before scintigraphy. The present study was undertaken to confirm this effect and to estimate the possible interference of various anti-inflammatory agents on the in vivo targeting of macrophages by J001X. Methylprednisolone, dexamethasone, indomethacin and methotrexate used at an immunosuppressive dose were tested to assess the possible risk of false-negative examinations in patients thus treated. Analysis of the results indicated that among the four drugs tested, only methylprednisolone at 0.5-1 mg/kg could interfere with J001X scintigraphy.