Approaches,levels and elements of vegetation research

The paper deals with the history of vegetation science viewed as a succession of paradigms. Alternative methodological approaches and alternative ways of viewing the elementary parts of vegetation at the coenotic level are replacing each other in the course of time. Vegetation research always involves a simplification of the vegetation objects by means of reduction and generalization. At any level there are certain traditional ways to describe the multiplicity of vegetation structure. According to the association approach, vegetation is mostly reduced to plant communities. The proponents of the continuum approach reduce the vegetation structure to the population level, and aggregate the populations in various ways. According to the ecosystem approach, various functional, more or less self-regulating vegetation elements can be described.     

Modeling Surface Growth of Escherichia coli on Agar Plates

Surface growth of Escherichia coli cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H.Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves.     

"Omics" data and levels of evidence for biomarker discovery

With the development of new technologies for assaying biological activity on a global basis in experimental samples, various new "-omics" signatures have been developed to predict disease progression. Such signatures hold the potential to alter the nature of clinical management of human disease. In this article, we describe some necessary statistical considerations needed to take these signatures from the discovery phase to a clinically useful assay. Much of the work discussed is in the area of cancer.     

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.     

A computer model for reshaping of cells in epithelia due to in-plane deformation and annealing

Although cell reshaping is fundamental to the mechanics of epithelia, technical barriers have prevented the methods of mechanics from being used to investigate it. These barriers have recently been overcome by the cell-based finite element formulation of Chen and Brodland. Here, parameters to describe the fabric of an epithelium in terms of cell shape and orientation and cell edge density are defined. Then, rectangular "patches" of model epithelia having various initial fabric parameters are generated and are either allowed to anneal or are subjected to one of several patterns of in-plane deformation. The simulations show that cell reshaping lags the deformation history, that it is allayed by cell rearrangement and that it causes the epithelium as a whole to exhibit viscoelastic mechanical properties. Equations to describe changes in cell shape due to annealing and in-plane deformation are presented.     

High-throughput characterization of lipopolysaccharide-binding proteins using mass spectrometry

Lipopolysaccharide (LPS)-binding proteins interact with LPS in human serum and mediate various immune responses. We describe a high-throughput LPS-binding protein profiling platform for discovering unknown LPS-binding proteins and potential inflammatory mediators. As a pull-down method, the LPS molecules were immobilized onto epoxy beads and then directly incubated with human serum to screen LPS-binding proteins. Through the "untargeted" mass spectrometric approach, potential LPS-binding proteins which elicit various immune responses in human serum were identified by a highly sensitive LTQ Orbitrap Hybrid Fourier Transform Mass Spectrometer (LTQ Orbitrap FT MS). Therefore, this mass spectrometry (MS)-based profiling method is straightforward for screening unknown LPS-binding proteins and provides physiologically relevant binding partners in human serum.     

A Computer Model for Reshaping of Cells in Epithelia Due to In-plane Deformation and Annealing

Although cell reshaping is fundamental to the mechanics of epithelia, technical barriers have prevented the methods of mechanics from being used to investigate it. These barriers have recently been overcome by the cell-based finite element formulation of Chen and Brodland. Here, parameters to describe the fabric of an epithelium in terms of cell shape and orientation and cell edge density are defined. Then, rectangular "patches" of model epithelia having various initial fabric parameters are generated and are either allowed to anneal or are subjected to one of several patterns of in-plane deformation. The simulations show that cell reshaping lags the deformation history, that it is allayed by cell rearrangement and that it causes the epithelium as a whole to exhibit viscoelastic mechanical properties. Equations to describe changes in cell shape due to annealing and in-plane deformation are presented.     

"Showing roughness in a beautiful way": talk about love, coercion, and rape in South African youth sexual culture

Sexual violence within as well as outside sexual relationships has far-reaching public health and human rights implications and is a continuing focus of popular debate, media coverage, and research in postapartheid South Africa. Partly because it has been shown to affect individual vulnerability to HIV/AIDS, sexual violence has in recent years become framed as a global public health issue. International research efforts to document the scale of this personally and politically sensitive problem can encounter conceptual, definitional, and methodological difficulties that anthropology is well placed to assist in alleviating. This article offers an ethnographic exploration of the spectrum of practices relating to sexual coercion and rape among young people in a township in the former Transkei region of South Africa. Contextualizing meanings of sexual coercion within local youth sexual culture, the article considers two emic categories associated with sex that is "forced": ukulala ngekani: "to sleep with by force" or ukunyanzela: "to force," both usually used to describe episodes occurring within sexual partnerships; and ukudlwengula, used to describe rape by a nonpartner or stranger. The article discusses the semantic content of and differences between these two key categories, demonstrating that encounters described as "forced sex" encompass not only various forms of sexual coercion but also, particularly in the narratives of young men, instances of more consensual sex. Of importance, in turn, in defining an act as "rape" rather than as "forced sex" are the character of the relationship between the two parties and interlinked ideas relating to exchange and sexual entitlement, love, and the importance of "intention," violation, and "deserving" victimhood.     

The real life of aged persons living alone in a depopulated area--in case of village a in Nagano Prefecture

Japan adopted a high economic growth policy and developed rapid industrialization since the 1960s. Consequently, Japanese have gotten various positive influences and negative influences. In this paper, we describe various positive influences and also we describe the "real life" of aged persons living alone in depopulated areas as one of the typical negative influences. For the purpose of investigating the actual lives of aged person living alone in depopulated areas, we carried out repeated interviews, research and direct observations of their work and lives in Village A. Village A is located in a mountain area 20 kilometers from Nagano City. The total population of the village was 3,294 in 1993, while it was 6,312 in 1960. The ratio is increasing rapidly year by year. In the village, the number of aged persons over 65 years old living alone, without any family, was 116 in 1994. The highest age is 94 years old. The number is increasing year by year. Through our study, it is made clear that aged persons over 65 years old living alone in the village have to manage everything in their daily lives by themselves to maintain their own safety, while accepting the influences of economic and industrial developments.     

Rocket-powered high-performance liquid chromatographic analysis of plant ascorbate and glutathione

We describe a robust procedure for the extraction and high-performance liquid chromatographic analysis of L-ascorbate (vitamin C), glutathione (gamma-glutamyl cysteinylglycine), and their respective oxidized forms from various plant tissues. Parameters such as the choice of extraction buffer, tissue disruption technique, sample stability, and separation conditions have all been optimized. In particular we found that the inclusion of the reducing agent dithiothreitol as a "stabilizer" in extracts with high phenolic content actually promoted oxidation of these antioxidants. Further, by using commercially available short "Rocket" HPLC columns in combination with high mobile-phase flow rates, analysis times were reduced to only 6min, making the method suitable for the high-resolution screening of large numbers of samples.     

"Real-time" monitoring of vibrissa contacts during rodent whisking

Rodent whisking behavior provides active touch as input into a widely studied model system of information processing and behavior. We previously developed a simple optoelectronic system to monitor whisker movements in "real time" in head held rats at rest or performing various tasks such as tactile discrimination. We now describe a simple piezioelectic film device for detecting initial whisker contacts during whisking also in real time. In some applications this is as effective as high-speed videos and can be configured to isolate the contacts from different whiskers. The construction of this simple device is detailed. In addition to providing information during recordings from awake animals, the device could be used, for example, as an operant "manipulandum" for contingent reinforcement of object detection with a whisker.     

Micro total analysis system (μ-TAS) in biotechnology

Nanobiotechnology raises fascinating possibilities for new analytical assays in various fields such as bioelectronic assembly, biomechanics and sampling techniques, as well as in chips or micromachined devices. Recently, nanotechnology has greatly impacted biotechnological research with its potential applications in smart devices that can operate at the level of molecular manipulation. Micro total analysis system (-TAS) offers the potential for highly efficient, simultaneous analysis of a large number of biologically important molecules in genomic, proteomic and metabolic studies. This review aims to describe the present state-of-the-art of microsystems for use in biotechnological research, medicine and diagnostics.     

Piezophysiology of yeast: occurrence and significance.

Hydrostatic pressure is a thermodynamic parameter that has recently received further consideration in various experimental fields. Although the physicochemical basis of pressure effects is well established, the effects of high pressure on in vivo biological processes have not been systematically investigated due to its presumed complexity. The word "piezophysiology" was proposed to describe the unique cellular responses to high pressure in living cells. The yeast Saccharomyces cerevisiae is one of the best-characterized organisms in many fields in bioscience. Here I review the accumulated literature on cellular responses to increasing hydrostatic pressure in yeast, focusing on survival, growth, gene expression and metabolism.     

Tumour markers in malignant diseases

The authors define the concept of "tumour markers" that indicate the presence of malignant diseases and describe their various stages of development. In addition, they demonstrate the classification, selection and clinical application of these markers. The theoretical and practical aspects of their clinical use are also discussed in terms of national and international expectations. Shortcomings in the clinical use of tumour markers in Hungary are touched upon and recommendations are offered for tumour marker development in this country.     

From protein structure to biochemical function?

Here we describe various methods currently under development aimed at identifying a proteins function from its three-dimensional structure. We are combining a number of these methods to create a pipeline of applications, called ProFunc, which will take a given 3D structure, run all the applications on it and compile and summarise the results obtained. The aim is to provide a best guess as to the proteins function from the evidence provided by the different methods. Here we present three examples, using structures solved by the Midwest Center for Structural Genomics consortium, illustrating the strengths and weaknesses of current approaches.     

Gene inactivation using the CRISPR/Cas9 system in the nematode <Emphasis Type="Italic">Pristionchus pacificus</Emphasis>

The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various –omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes.     

Purification of DNA from agarose gels

Summary We describe a rapid and easily reproducible modification of the freeze-squeeze method of separating DNA from agarose gels. Our method involves slicing out the agarose gel portion which contains the DNA of interest, freezing this gel slice at –20°C, then centrifuging the frozen slice in a filtration unit which contains a cellulose acetate filter. The agarose is retained on the filter and the filtrate contains the DNA. DNA purified in this manner could be completely digested with restriction endonucleases and completely ligated with DNA ligase, without further purification. The percentages of recovery for various sizes of linear and plasmid double-stranded DNA ranged from 57 to 69%. The procedure takes less than 30 minutes to perform.     

Gateway-compatible vectors for plant functional genomics and proteomics

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.     

A structural study of interfacial phospholipid and lung surfactant layers by transmission electron microscopy after Blodgett sampling: influence of surface pressure and temperature.

Monolayer studies of the lung surfactant extract (LSE), dipalmitoyl phosphatidilcholine (DPPC) and dioleyl phosphatidilcholine (DOPC) have been performed in the dynamic condition at various temperatures. These compounds were also studied by differential scanning calorimetry, and the Langmuir Blodgett films were examined by electron microscopy. The combination of these techniques allowed us to describe precisely the collapse process, which was found to be different above and below the transition temperature of the lipids. However, whereas a phase separation for DPPC/DOPC mixtures occurred at all temperatures studied, this separation was observed for LSE only at temperatures lower than that characteristic of the "rigid state" to "liquid-like state" transition temperature. The ability of LSE to rapidly respread upon decompression appears to be due to the formation of piled amorphous aggregates formed during compression of its monolayers.