Parallel Sequence Matching with TACO's Distributed Object Groups – A Case Study from Molecular Biology

TACO is a template library that implements higher-order parallel operations on distributed object sets by means of reusable topology classes and C++ function templates. In this paper we discuss an experimental application that exploits TACO's distributed object groups and collective operations for computing the similarity between groups of molecular sequences, a computationally intensive core problem in molecular biology research. In particular we show how TACO's distributed collections can be conveniently combined with well known concepts found in the C++ standard template library (STL) to solve matching and sorting problems effectively on distributed hardware platforms. The resulting implementation is concise and gives excellent parallel performance on PC- and workstation clusters.     

A sticker-based model for DNA computation.

We introduce a new model of molecular computation that we call the sticker model. Like many previous proposals it makes use of DNA strands as the physical substrate in which information is represented and of separation by hybridization as a central mechanism. However, unlike previous models, the stickers model has a random access memory that requires no strand extension and uses no enzymes; also (at least in theory), its materials are reusable. The paper describes computation under the stickers model and discusses possible means for physically implementing each operation. Finally, we go on to propose a specific machine architecture for implementing the stickers model as a microprocessor-controlled parallel robotic workstation. In the course of this development a number of previous general concerns about molecular computation (Smith, 1996; Hartmanis, 1995; Linial et al., 1995) are addressed. First, it is clear that general-purpose algorithms can be implemented by DNA-based computers, potentially solving a wide class of search problems. Second, we find that there are challenging problems, for which only modest volumes of DNA should suffice. Third, we demonstrate that the formation and breaking of covalent bonds is not intrinsic to DNA-based computation. Fourth, we show that a single essential biotechnology, sequence-specific separation, suffices for constructing a general-purpose molecular computer. Concerns about errors in this separation operation and means to reduce them are addressed elsewhere (Karp et al., 1995; Roweis and Winfree, 1999). Despite these encouraging theoretical advances, we emphasize that substantial engineering challenges remain at almost all stages and that the ultimate success or failure of DNA computing will certainly depend on whether these challenges can be met in laboratory investigations.     

Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena.

Cellular events are accomplished by the coordinated interactions of cellular components within the three-dimensional context of a cell. Simultaneous observation of multiple components in three dimensions can be essential for understanding such interactions. Toward this end, we have developed a computerized microscope workstation capable of recording three-dimensional images of multiple cellular components in fixed and living cells. All aspects of microscope control, data collection, image processing and analysis can be performed on the one workstation. In this report, we describe the components and capabilities of this integrated system. In addition, we discuss some general problems of multiple-wavelength, three-dimensional imaging and our application of this technology to the analysis of chromosome organization in Drosophila melanogaster. Three-dimensional imaging of fixed embryos stained by indirect immunofluorescence has revealed the structural organization of chromosomes, microtubules, and the nuclear lamins. Imaging of living embryos injected with fluorescently labelled proteins has confirmed and extended these results by allowing the study of these structures throughout the cell cycle. The combination of the molecular specificity of fluorescence microscopy and the three-dimensional structural information obtained by our workstation has provided novel insights into the dynamic aspects of chromosome behavior during the cell cycle. We believe this system has many important applications in the study of the molecular basis of cellular events.     

The future of aging therapies

Advances in understanding aging processes and their consequences are leading to the development of therapies to slow or reverse adverse changes formerly considered to be "normal" aging and processes that underlie multiple age-related conditions. Estimating the effectiveness of candidate aging therapies, whose effects on human aging may require many years to determine, is a particular challenge. Strategies for identifying candidate interventions can be developed through multiple approaches, including the screening of molecular targets and pathways in vitro and in animal models, informed as well by evidence from human genetic and epidemiologic data. A number of recently established programs and networks can serve as resources for such research. For all these research approaches, from in vitro molecular studies to clinical trials, contributions of cell and molecular biology are crucial and offer the prospect of therapeutic advances that address fundamental biological processes as well as the clinically important challenges of aging.     

Signaling involved in stem cell reprogramming and differentiation

Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have revealed that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell programming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review, the molecular interactions and signaling pathways related to stem cell differentiation are discussed.     

Programmable ribozymes for mischarging tRNA with nonnatural amino acids and their applications to translation

Here we describe a novel technology that allows users to charge nonnatural amino acids onto any tRNA. This technology is based on a resin-immobilized ribozyme system, called Flexiresin. It enables users to readily and rapidly synthesize misacylated tRNAs with a wide variety of phenylalanine analogs. Since Flexiresin is reusable and little effort is necessary for regeneration, it is economical and convenient. Moreover, it can adapt to virtually any tRNA chosen by the user, and can therefore be applied to not only a single site mutation but also multiple sites with designated nonnatural amino acids when both the amber and programmed frame-shift mutations are utilized. The original ribozyme utilized for Flexiresin was artificially generated in vitro, and thus the technology in principle could be broadened from Phe analogues to essentially any amino acid.     

Molecular imaging of cell-based cancer immunotherapy

Cell-based cancer immunotherapy represents a new and powerful weapon in the arsenal of anticancer treatments. Non-invasive monitoring of the disposition, migration and destination of therapeutic cells will facilitate the development of cell based therapy. The therapeutic cells can be modified intrinsically by a reporter gene or labeled extrinsically by introducing imaging probes into the cells or on the cell surface before transplant. Various advanced non-invasive molecular imaging techniques are playing important roles in optimizing cellular therapy by tracking cells and monitoring the therapeutic effects of transplanted cells in vivo. This review will summarize the application of multiple molecular imaging modalities in cell-based cancer immunotherapy.     

An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.     

Molecular pathology and targeted therapy of clear cell renal cancer

The golden standard of care for advanced renal cell cancer (RCC) was until now the cytokine therapy with relatively low response rates. Advances of molecular genetics in RCC revealed several molecular targets such as VHL and angiogenic genotype or EGFR in the clear cell variant. Among the novel targeted agents, multiple tyrosine kinase inhibitors were proved to be clinically effective against advanced clear cell renal cancer, changing the standard of care. It is a further question how molecular diagnostics can improve these results by the detection of these targets or gene defects in individual tumors.     

Utilization of adenovirus vectors for multiple gene transfer applications

Mammalian viruses have evolved over millions of years to achieve a single goal, namely to rapidly enter a host mammalian cell, in order to achieve virus propagation. In so doing, these biologic parasites have acquired the molecular tools to rapidly and efficiently deliver their own nucleic acids into the nucleus of the host cell. The human adenovirus is one of the best studied of these parasites. As such the adenovirus has been re-engineered to allow it to be used as a tool to allow researchers to deliver desired nucleic acid sequences into a large variety of cell targets, both in tissue culture systems, as well as directly into living animals. Adenovirus based gene transfer systems can overcome most of the problems inherent to high efficiency gene transfer, and perform in a fashion that in many ways cannot be matched by most other currently utilized gene transfer systems. This article will attempt to summarize the multiple attributes of this widely utilized gene delivery system.     

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.     

DNA在鸟类分子系统发育研究中的应用

鸟类分子系统发育研究中常用的DNA技术有DNA杂交、RFLP和DNA序列分析等。DNA杂交技术曾在鸟类中有过大规模的应用,并由此诞生了一套新的鸟类分类系统。在鸟类的RFLP分析中,用的最多的靶序列是线粒体DNA。DNA序列分析技术被认为是进行分子系统发育研究最有效、最可靠的方法。在DNA序列分析中,线粒体基因应用最广泛,但由于其自身的一些不足,近年来,不少学者把目光投向了核基因,将线粒体基因和核基因结合起来进行系统发育研究。目前在鸟类分子系统发育中,应用较多的核基因是scnDNA,其内含子可以用于中等阶元水平的系统研究,而外显子主要用于高等阶元的系统研究。除了分子标记自身的问题之外,鸟类分子系统发育研究中还存在着方法上的问题,包括分子标记的选择,样本数量以及数据处理等。今后鸟类分子系统发育研究应该更加注重方法的标准化。     

Protocol for the fabrication of enzymatically crosslinked gelatin microchannels for microfluidic cell culture

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. By producing reusable poly(dimethyl siloxane) molds using standard photolithography, gelatin can be molded into microchannel geometries. The gelatin is crosslinked with the naturally occurring enzyme transglutaminase via a straightforward process that can produce devices suitable for cell culture. The protocol takes approximately 1 day from the start of gelatin preparation to cell seeding. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.     

Eco-efficiency of disposable and reusable surgical instruments—a scissors case

Purpose

In recent years, the rising costs and infection control lead to an increasing use of disposable surgical instruments in daily hospital practices. Environmental impacts have risen as a result across the life cycle of plastic or stainless steel disposables. Compared with the conventional reusable products, different qualities and quantities of disposable scissors have to be taken into account. An eco-efficiency analysis can shed some light for the potential contribution of those products towards a sustainable development.

Methods

Disposable scissors made of either stainless steel or fibre-reinforced plastic were compared with reusable stainless steel scissors for 4,500 use cycles of surgical scissors used in Germany. A screening life cycle assessment (LCA) and a life cycle costing were performed by following ISO 14040 procedure and total cost of ownership (TCO) from a customer perspective, respectively. Subsequently, their results were used to conduct an eco-efficiency analysis.

Results and discussion

The screening LCA showed a clear ranking regarding the environmental impacts of the three types of scissors. The impacts of the disposable steel product exceeds those of the two others by 80 % (disposable plastic scissors) and 99 % (reusable steel scissors), respectively. Differences in TCO were smaller, however, revealing significant economic advantages of the reusable stainless steel product under the constraints and assumptions of this case study. Accordingly, the reusable stainless steel product was revealed as the most eco-efficient choice. It was followed by the plastic scissors which turned out to be significantly more environmentally sound than the disposable stainless steel scissors but also more cost-intensive.

Conclusions

The overall results of the study prove to be robust against variations of critical parameters for the prescribed case study. The sensitivity analyses were also conducted for LCA and TCO results. LCA results are shown to be reliable throughout all assumptions and data uncertainties. TCO results are more dependent on the choice of case study parameters whereby the price of the disposable products can severely influence the comparison of the stainless steel and the plastic scissors. The costs related to the sterilisation of the reusable product are strongly case-specific and can reduce the economic benefit of the reusable scissors to zero. Differences in environmental and economic break-even analyses underline the comparatively high share of externalised environmental costs in the case of the disposable steel product.     

Timing cell-fate determination during asymmetric cell divisions

From invertebrates to mammals, cell-cycle progression during an asymmetric cell division is accompanied by precisely timed redistribution of cell-fate determinants so that they segregate asymmetrically to enable the two daughter cells to choose different fates. Interestingly, studies on how cell fates are specified in such divisions reveal that the same fate determinants can be reiteratively used to specify a variety of cell types through multiple rounds of cell divisions or to exert seemingly contradictory effects on cell proliferation and differentiation. Here I summarize the molecular mechanisms governing asymmetric cell division and review recent findings pointing to a novel mechanism for coupling intracellular signaling and cell-cycle progression. This mechanism uses changes in the morphology, subcellular distribution, and molecular composition of cellular organelles like the Golgi apparatus and centrosomes, which not only accompany the progression of cell cycle to activate but also temporally constrain the activity of fate determinants during asymmetric cell divisions.     

Equilibrium expert: an add-in to Microsoft Excel for multiple binding equilibrium simulations and parameter estimations

An add-in to Microsoft Excel was developed to simulate multiple binding equilibriums. A partition function, readily written even when the equilibrium is complex, describes the experimental system. It involves the concentrations of the different free molecular species and of the different complexes present in the experiment. As a result, the software is not restricted to a series of predefined experimental setups but can handle a large variety of problems involving up to nine independent molecular species. Binding parameters are estimated by nonlinear least-square fitting of experimental measurements as supplied by the user. The fitting process allows user-defined weighting of the experimental data. The flexibility of the software and the way it may be used to describe common experimental situations and to deal with usual problems such as tracer reactivity or nonspecific binding is demonstrated by a few examples. The software is available free of charge upon request.     

Designing a reusable system based on nanodiamonds for biochemical determination of urea

A reusable system including urease covalently bound to the surface of modified nanodiamonds (MNDs) has been developed for the multiple determination of urea. The immobilized enzyme exhibits functional activity and catalyzes the hydrolysis of urea to yield ammonia. The presence of ammonia is confirmed by the formation of a colored product after the addition of chemical reagents. It was shown that the MNDs-urease complex can function in a wide range of temperatures and pH as well as in deionized water. The complex provides a linear yield of the product at low analyte concentrations and allows the multiple determination of urea in vitro.