Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

A simple and fast method for cell recovery and DNA content analysis from various mouse tissues by flow cytometry

Cell division in tissues can be investigated in various ways. We present here a method for improving cell recovery and cell cycle analysis for a wide range of mouse tissues. This strategy combines a cell isolation procedure for various mouse tissues based on intracardiac perfusion and subsequent treatment followed by flow cytometry. This easy and reproducible method allows a rapid analysis of nuclear DNA content, providing an estimate of the cell number at different phases of the cell cycle. This combined procedure could also be used for the isolation of specific cell subpopulations from different mouse tissues by fluorescence activated cell sorting.

Individual variability of behavioural responses by Wandering Albatrosses (Diomedea exulans) to human disturbance

Sub-Antarctic Marion Island has had a permanent research station for 50 years and the islands Wandering Albatrosses have been intensively studied for 20 years. The reactions of breeding birds to approaches by a human on foot were recorded. Three response variables were calculated: intensity of vocal reaction (IVR), intensity of non-vocal reaction (INR) and overall response index (ORI). At 5 m from the nest, twice as many birds stood and/or vocalised as at 15 m. Nearest neighbour distance, age and gender did not explain individual variability of responses. Study colony birds had higher IVR scores than non-study colony birds; birds at colonies closest to the station had the highest ORI scores. A better breeding record was associated with lower IVR and ORI scores, but a causative relationship remains to be demonstrated. A minimum viewing distance of 25 m is recommended for breeding Wandering Albatrosses.

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Real-time Investigation of SV40 Large T-antigen Helicase Activity Using Surface Plasmon Resonance

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl-CoA in <Emphasis Type="Italic">Methanosarcina thermophila</Emphasis>

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates. Electronic Supplementary Material  Supplementary material is available for this article at

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Hierarchical analysis of piecewise affine models of gene regulatory networks

A key point in the analysis of dynamical models of biological systems is to handle systems of relatively high dimensions. In the present paper we propose a method to hierarchically organize a certain type of piecewise affine (PWA) differential systems. This specific class of systems has been extensively studied for the past few years, as it provides a good framework to model gene regulatory networks. The method, shown on several examples, allows a qualitative analysis of the asymptotic behavior of a PWA system, decomposing it into several smaller subsystems. This technique, based on the well-known strongly connected components decomposition, is not new. However, its adaptation to the non-smooth PWA differential equations turns out to be quite relevant because of the strong discrete structure underlying these equations. Its biological relevance is shown on a 7-dimensional PWA system modeling the gene network responsible for the carbon starvation response in Escherichia coli.

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Time-sharing parallel applications through performance-targeted feedback-controlled real-time scheduling

Most parallel machines, such as clusters, are space-shared in order to isolate batch parallel applications from each other and optimize their performance. However, this leads to low utilization or potentially long waiting times. We propose a self-adaptive approach to time-sharing such machines that provides isolation and allows the execution rate of an application to be tightly controlled by the administrator. Our approach combines a periodic real-time scheduler on each node with a global feedback-based control system that governs the local schedulers. We have developed an online system that implements our approach. The system takes as input a target execution rate for each application, and automatically and continuously adjusts the applications’ real-time schedules to achieve those rates with proportional CPU utilization. Target rates can be dynamically adjusted. Applications are performance-isolated from each other and from other work that is not using our system. We present an extensive evaluation that shows that the system remains stable with low response times, and that our focus on CPU isolation and control does not come at the significant expense of network I/O, disk I/O, or memory isolation.

Leitlinien zur molekulargenetischen Diagnostik: Fragiles-X und Fragiles-X assoziiertes Tremor/Ataxie Syndrom

Ohne Zusammenfassung

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Hennigian Phylogenetic Systematics and the ‘Groundplan’ vs. ‘Post-Groundplan’ Approaches: A Reply to Kukalová-Peck

The recent contribution by Jarmila Kukalová-Peck on Hennigian phylogenetics and hexapod limb evolution is critically evaluated.

Causal Explanation of Indonesian Forest Fires: Concepts, Applications, and Research Priorities

Problems with studies, claims, and assumptions that have been made about the causes of fires in Indonesia's tropical moist forests are identified, and the kinds of concepts, methods, prioritizing, and data needed to resolve the problems are discussed. Separate sections are devoted to studying ignitions, studying fire susceptibility and fire behavior, and using the goal of causal explanation to guide interdisciplinary research.

The connectionist framework for gene regulation

I show that gene regulation networks are qualitatively consistent and therefore sufficiently similar to linearly seperable connectionist networks to warrant that the connectionist framework be applied to gene regulation. On this view, natural selection designs gene regulation networks to overcome the difficulty of development. I offer some general lessons about their evolvability that can be learned by examining the generic features of connectionist networks.

Face to Face

Over evolutionary time, humans have developed a selective sensitivity to features in the human face that convey information on sex, age, emotions, and intentions. This ability might not only be applied to our conspecifics nowadays, but also to other living objects (i.e., animals) and even to artificial structures, such as cars. To investigate this possibility, we asked people to report the characteristics, emotions, personality traits, and attitudes they attribute to car fronts, and we used geometric morphometrics (GM) and multivariate statistical methods to determine and visualize the corresponding shape information. Automotive features and proportions are found to covary with trait perception in a manner similar to that found with human faces. Emerging analogies are discussed. This study should have implications for both our understanding of our prehistoric psyche and its interrelation with the modern world.

Objects limit human comprehension

This paper demonstrates that the human visual system, the primary sensory conduit for primates, processes ambient energy in a way that obligatorily constructs the objects that we ineluctably perceive. And since our perceptual apparatus processes information only in terms of objects (along with the properties and movements of objects), we are limited in our ability to comprehend ‘what is’ when we move beyond our ordinary world of midsize objects—as, for example, when we address the micro microworld of quantum physics.

Multiple realization in comparative perspective

Arguments for multiple realization depend on the idea that the same kind of function is realized by different kinds of structures. It is important to such arguments that we know the kinds used in the arguments have been individuated properly. In the philosophical literature, though, claims about how to individuate kinds are frequently decided on intuitive grounds. This paper criticizes this way of approaching kinds by considering how practicing researchers think about the matter. I will consider several examples in which the practice of researchers on comparative vision conflicts with the standard account of these issues.

The Evolution of Complex Organs

The origin of complex biological structures has long been a subject of interest and debate. Two centuries ago, natural explanations for their occurrence were considered inconceivable. However, 150 years of scientific investigation have yielded a conceptual framework, abundant data, and a range of analytical tools capable of addressing this question. This article reviews the various direct and indirect evolutionary processes that contribute to the origins of complex organs. The evolution of eyes is used as a case study to illustrate these concepts, and several of the most common misconceptions about complex organ evolution are discussed.