Possibility for the Conjugated Use of Photodynamic Therapy and Electrosurgical Devices

Because tissue optics limits the treated volume during anti-tumor Photodynamic Therapy (PDT), its conjugation with prior tissue debulking has been suggested clinically. In this context, the conjugation of radiofrequency ablation and PDT has already been demonstrated. However, the basic principles that enable the success of these protocols have not been discussed. This proof-of-principle study analyzes the possibility of conjugating electrosurgery (ES) and PDT, analyzing different sequences of photosensitizer (PS) administration in an animal model. The animals were distributed over five groups: ES, PS+Light, PS+ES, ES+PS+Light and PS+ES+Light. The PS Photogem was administered systemically. An electrosurgical unit (480 kHz) was used to remove a portion of the liver, leaving a plane surface for PDT illumination (630 nm, 150 J/cm²). Fluorescence was collected during the stages of the experiment to monitor the PS accumulation. After 30 hours, histological processing was performed. The fluorescence spectra revealed strong Photogem emission in both administration sequences (ES+PS; PS+ES), and little PS bleach after ES was observed. The maximum necrosis depth was observed for the PS+ES+Light group—(716 ± 75) μm—higher than its respective control group (160 ± 28) μm, proving successful conjugation. Histological features from ES and PDT on both conjugation sequences were observed. Pre-photosensitized tissue presented decreased ES-related thermal damage. A simple physical hypothesis, based on the Joule effect and the tissue electrical conductivity, was proposed to support these findings. In conclusion, the results successfully demonstrated the possibility of conjugating ES and PDT in a single protocol.     

A Simple Flow Indicator for Use in Perfusion Fixation

In perfusion fixation, we have preferred gravity flow rather than a peristaltic forced flow because the latter may cause rupture of small blood vessels. One difficulty encountered in the gravity flow method is detection of flow stoppage, often caused by the formation of a blood clot. If this is apparent soon enough, steps can be taken to re-establish the flow, Flow stoppage is not usually detected for several minutes—resulting in poor fixation of the organ involved. To allow immediate detection of stoppage, a glass flow indicator (figure 1) was manufactured     

The Use of the Microcomposter to Study the Dynamics of a Mini-Ecosystem

In this activity, students learn about the important topic of invasive species, specifically Zebra Mussels. Students role-play different characters in a real-life situation: the trial of the Zebra Mussel for unlawful disruption of the Great Lakes ecosystem. Students will also learn about jurisprudential inquiry by examining the trial process. This activity will reinforce important knowledge and skills underscored in the Life Science and Science in Personal and Social Perspectives Standards in the National Science Education Standards (National Research Council 1996).     

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI     

Evaluation of Six Fluorescent Protein Stains for Use in Flow Microfluorometry

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluoresoein isothiocyanate (FITC) and rho-damine B isothiocyanate (RITC). Both fluoreseamine and 8-aniline-1-naphthakne sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) WCR found unsatisfactory in these studies, since the former dye tended to diffuse from the all., while the latter induced excessive all clumping and cell loss. These techniques have application to immunofluoregcence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.     

Evaluation of Six Fluorescent Protein Stains for Use in Flow Microfluorometry

Flow microfluorometric (FMF) analysis of stained cells has provided protein distribution histograms for large populations of cells. Spectral data and staining protocols were evaluated for six fluorescent protein dyes suggested for staining cells in liquid suspension. The requirements for dyes and/or staining protocol included minimal cell clumping and cell loss, near-optimal dye excitation at existing laser wavelengths, and tenacity of the dye/protein interaction. These criteria were best satisfied by fluoresoein isothiocyanate (FITC) and rho-damine B isothiocyanate (RITC). Both fluoreseamine and 8-aniline-1-naphthakne sulfonic acid (ANSA) showed potential applicability for use in systems where excitation wavelengths in the ultraviolet range are available. Protein staining with fluorescamine was extremely rapid. Brilliant sulfaflavine and 1-dimethyl-aminonaphthalene-5-sulfonyl chloride (DANSYL) WCR found unsatisfactory in these studies, since the former dye tended to diffuse from the all., while the latter induced excessive all clumping and cell loss. These techniques have application to immunofluoregcence analysis and can also be profitably employed in dual-parameter analysis systems in connection with double-staining techniques for simultaneous DNA and protein analysis.     

Experience with the Use of Substance Flow Analysis in Denmark

Substance flow analysis (SFA) has been used by the Danish Environmental Protection Agency for more than two decades to identify sources of hazardous substance releases to the environment and to waste streams. More than 35 SFAs have been undertaken using a unified methodology. This article discusses key elements of the methodology and application of the results of the SFAs in Denmark, illustrated by examples. The use of repeated SFAs in the evolution of substance regulation is demonstrated for lead. A cadmium SFA illustrates the use of SFAs to monitor changes in consumption and release of a hazardous substance. Estimates of sources are presented for a number of toxic and resource-intensive metals in solid waste incineration and sewage treatment. Most of the SFAs concern hazardous substances, but aluminum represents a case in which the resource perspective is an important element in a national SFA. The Danish SFA on brominated flame retardants illustrates some methodological problems with conducting national SFAs in a global economy in which the substances are mainly imported in finished products.     

小麦亲本间遗传距离预测杂种优势可行性的研究

本文通过10个小麦品种和按双列杂交配制的45个组合为材料,研究亲本间遗传距离预测杂种优势的可行性。结果表明：93.02％的样本实测值落在预测区间。     

Development and Use of Flow Cytometry for Detection of Airborne Fungi

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.     

狂犬病病毒Vero细胞适应株的建立及其应用于疫苗生产的可行性探讨

将狂犬病病毒７ａＧ固定毒适应于Ｖｅｒｏ细胞,建立了稳定的ＶＲＧ适应株。病毒滴度可达７．６９ｌｏｇＬＤ５０／ｍｌ,病毒最适增殖时间５—７天,最适种毒比例１：１０￣３。应用转瓶培养Ｖｅｒｏ细胞增殖病毒,收获病毒液经β—丙内脂灭活,超离浓缩制备疫苗,效力试验结果（ＮＩＨ法）较原制疫苗成倍增高,且都大于２．５ＩＵ／２ｍｌ,表明ＶＲＧ适应株可应用于制备Ｖｅｒｏ细胞狂犬病疫苗。     

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.     

Toward the Use of Genomics to Study Microevolutionary Change in Bacteria

Bacteria evolve rapidly in response to the environment they encounter. Some environmental changes are experienced numerous times by bacteria from the same population, providing an opportunity to dissect the genetic basis of adaptive evolution. Here I discuss two examples in which the patterns of rapid change provide insight into medically important bacterial phenotypes, namely immune escape by Neisseria meningitidis and host specificity of Campylobacter jejuni. Genomic analysis of populations of bacteria from these species holds great promise but requires appropriate concepts and statistical tools.Bacteria lack a natural reproductive system, comparable to meiosis in eukaryotes, that segregates genes randomly. Instead, they evolve progressively through mostly small genetic changes, a proportion of which have noteworthy phenotypic effects. Some phenotypes are intrinsically difficult to study in the laboratory: virulence in humans or adaptation to particular ecological niches, for example. For these traits in particular, a promising avenue for scientific investigation is to identify the genetic changes that have provided the basis for their evolution in natural populations.Most human phenotypes are hard to study in vitro and, consequently, methods for relating differences amongst humans to natural genetic variation are well developed. Association studies were proposed as an effective way of identifying genes with small phenotypic effects more than a decade ago [1] and, although initially controversial [2], the recent development of arrays for genotyping hundreds of thousands of single nucleotide polymorphisms (SNPs) scattered across the whole genome has allowed the approach to be successfully applied to many different human diseases and other phenotypes [3]. This success should inspire the development of equivalent protocols within bacteriology.One challenge in developing generally applicable protocols for mapping phenotypic traits in bacteria is that processes by which microevolution occurs vary tremendously between species. For example, the human pathogen Mycobacterium tuberculosis, the causal agent of tuberculosis (TB), diverged recently from an obscure organism occasionally isolated from humans in Africa called Mycobacterium canetti [4]. M. tuberculosis shows very little variation and there is no evidence of strains acquiring DNA by import from other M. tuberculosis strains or indeed from any other organism, so that individuals are clones of each other, distinguished only by rare mutations or other small changes. By contrast, individual Helicobacter pylori, a cause of gastric cancer, acquire DNA from other members of the species at an extremely high rate. Consequently, as well as varying in gene content [5], strains isolated from different host individuals in the same ethnic group typically differ from each other at approximately 3% of nucleotides in core genes, and this diversity segregates nearly randomly [6]. The majority of bacterial species fall between these extremes, with their genomes showing signs of both clonal descent and DNA import from other strains.In this essay, I will argue that the clonal mode of reproduction shared by all bacteria and Archaea, in which replication occurs by binary fission, in fact provides an extremely powerful context for association studies. These studies will require both appropriate technologies for genotyping and evolutionary analysis and judiciously chosen strain collections. I will here concentrate on two examples in which placing evolutionary changes in their clonal context provides the power to relate phenotype to genotype. Population-scale genome sequencing promises to allow a full and unbiased catalogue of variation within the same clonal context. This reconstruction will facilitate identification of loci that show correlations with phenotype or anomalous patterns that indicate natural selection, with minimal assumptions about the mechanisms by which phenotypes change.     

On the Use of a Simple Physical System Analogy to Study Robustness Features in Animal Sciences

Environmental perturbations can affect the health, welfare, and fitness of animals. Being able to characterize and phenotype adaptive capacity is therefore of growing scientific concern in animal ecology and in animal production sciences. Terms borrowed from physics are commonly used to describe adaptive responses of animals facing an environmental perturbation, but no quantitative characterization of these responses has been made. Modeling the dynamic responses to an acute challenge was used in this study to facilitate the characterization of adaptive capacity and therefore robustness. A simple model based on a spring and damper was developed to simulate the dynamic responses of animals facing an acute challenge. The parameters characterizing the spring and the damper can be interpreted in terms of stiffness and resistance to the change of the system. The model was tested on physiological and behavioral responses of rainbow trout facing an acute confinement challenge. The model has proven to properly fit the different responses measured in this study and to quantitatively describe the different temporal patterns for each statistical individual in the study. It provides therefore a new way to explicitly describe, analyze and compare responses of individuals facing an acute perturbation. This study suggests that such physical models may be usefully applied to characterize robustness in many other biological systems.     

Optimisation of a Stirred Bioreactor through the Use of a Novel Holographic Correlation Velocimetry Flow Measurement Technique

We describe a method for measuring three dimensional (3D) velocity fields of a fluid at high speed, by combining a correlation-based approach with in-line holography. While this method utilizes tracer particles contained within the flow, our method does not require the holographic reconstruction of 3D images. The direct flow reconstruction approach developed here allows for measurements at seeding densities in excess of the allowable levels for techniques based on image or particle reconstruction, thus making it suited for biological flow measurement, such as the flow in bioreactor. We outline the theory behind our method, which we term Holographic Correlation Velocimetry (HCV), and subsequently apply it to both synthetic and laboratory data. Moreover, because the system is based on in-line holography, it is very efficient with regard to the use of light, as it does not rely on side scattering. This efficiency could be utilized to create a very high quality system at a modest cost. Alternatively, this efficiency makes the system appropriate for high-speed flows and low exposure times, which is essential for imaging dynamic systems.     

Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.     

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system¹. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury^2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes⁴. This method focuses on neutrophil transmigration across human endothelial cells.One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)⁵. Neutrophil isolation has been described visually elsewhere⁶; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression⁷. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature^7,8.The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro^9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.     

Possibility to use the lyophilization for the prolongation of the stability of biological compounds used in cosmetic production

The investigations were conducted with using aquatic hydrolysate of protein 5%, 10%, 20% and 50%. Starting product to prepare solutions with different concentrations, exposed on lyophilisation was pork gelatin (Polonaise, Belgium). The aim of investigations was to determine on effect of lyophilization process on the microbiological and physico-chemistral stability of protein hydrolisate. Physical parameters of lyophilization of protein hydrolysate solutions of different concentrations and physical properties of the lyophilized hydrolysates were determined. Microbiological analysis demonstrated that lyophilization protected the hydrolysates stored at 4 degrees C and 22 degrees C for 60 or 100 days against bacterial contamination. On the other hand, the inhibition of the bacterial multiplication in nonlyophilized hydrolysates was obtained only during storage at -20 degrees C. A proper structure and good dissolubility of the lyophilizated of preparation were obtained for 5%, 10% and 20% hydrolisate and for of the maximum kinematical viscosity 2923 mm2/s.