Bostrycin, a novel coupling agent for protein immobilization and prevention of biomaterial-centered infection produced by Nigrospora sp. No. 407

Bostrycin, a red antibacterial agent with tetrahydroanthraquinone structure, has been isolated from Nigrospora sp. No. 407. This study investigated the potential antibacterial and multifunctional properties of matrixes through immobilization of bostrycin on their surface for immobilization of protein and prevention of bacterial growth. Bostrycin was immobilized on nonwoven polypropylene (PP) fabric by a technique using glutaraldehyde and polyethyleneimine for the activation of the surface. Glucose oxidase immobilized on bostrycin-treated nonwoven PP fabric showed high activity. The immobilization process improved thermal stability of the enzymes. During repeated assay for 30 cycles, the enzyme activity dropped to only 70% of the initial activity. Both bostrycin-treated nonwoven PP fabric sample and subsequently immobilized glucose oxidase sample on the surface also still exhibited a bacteriostatic effect. This is the first study to show that bostrycin is a promising coupling agent for surface modification on matrix and its potential applications in protein immobilization and biomaterial-centered infection.     

Computerised topographical mapping of scalp recorded event-related potentials

A technique is described for the recording of multichannel cerebral evoked potential data and their conversion to colour-coded displays of scalp electrical activity at up to 40 instants in time. Such mapping gives a more readily assimilated picture of the evolution of the potential changes over the scalp than the raw waveforms. The method employs a PDP 11/23 computer for data collection and surface topography calculation, and a Tektronix 4662 digital plotter with 8 colour option for the display of spatio-temporal maps.     

Spatio-temporal mapping of evoked cerebral activity

A technique is described for the colour-coded display of averaged scalp electrical activity at 40 instants in time. An application of this technique to the pattern reversal visual evoked potential is discussed, showing the value of spatio-temporal mapping in the interpretation of multichannel evoked potential recordings.     

耗竭性游泳对大鼠心肌线粒体膜功能的影响

耗竭性游泳对大鼠心肌线粒体膜功能的影响王文信,丁树哲,许豪文（华东师范大学体育系运动生化实验室,上海２０００６２）关键词心肌线粒体,内膜表面电位,游离钙,总钙,合成活力长时间耗竭性游泳或跑步引起心肌、骨骼肌、肝脏等线粒体结构和功能变化,这些变化可能与...     

In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity.     

Surface replicas of pulmonary endothelial cells in culture.

Pulmonary endothelial cells possess a variety of enzymes on their surface. However, the topographical distribution of these enzymes is not known. In this report we describe a simple technique for the preparation of surface replicas of pulmonary endothelial cells using an unmodified critical point drying apparatus and a high vacuum freeze-etch unit. The technique should be applicable for use with all cells in monolayer culture. Endothelial cells grown on glass slides or coverslips were washed thoroughly, fixed and dehydrated. The monolayers were then critical point dried. Surface replicas were prepared in a Balzer's freeze-etch unit. The advantages of surface replicas are that they can be examined with the resolving power of the transmission electron microscope and can be used to examine whole cells. The potential for the surface replica technique may lie in mapping specific enzymes, receptors and cell surface factors. Therefore, to provide a basis for such studies, we have described the appearance of pulmonary endothelial cells in surface replicas.     

The contribution of nitrification in the water column and profundal sediments to the total oxygen deficit of the hypolimnion of a mesotrophic lake (Grasmere,English Lake District)

Estimates ofin situ nitrifying activity have been made in the hypolimnetic water column and surface 1.0 cm of profundal sediments at 2 sites in Grasmere, a mesotrophic lake in the English Lake District. Increases of nitrate concentrations were used to estimate nitrification in the water column whereas a mini-core technique, involving the use of a nitrification inhibitor (allylthiourea), was used to estimate the rate in surface sediments. The pattern of oxygen depletion in the water column was used to estimate the maximum depth to which sediments affect the overlying water. Nitrification in the sediment and in the water column made approximately equal contributions to the total areal oxygen deficit and, as a whole, nitrification accounted for 15–20% of the total oxygen depletion. There was no significant difference in oxygen depletion due to nitrification between the 2 sites. Attempts were made, using the nitrification potential technique, to determine the depth distribution of nitrifying activity in the surface 1.0 cm of sediment.     

Multiple labeling of cellular constituents by combining surface reflection interference and fluorescence microscopy

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.     

Demonstration of biofilm removal from type 304 stainless steel using pulsed-waveform electropolishing

Abstract

This article describes an electrochemical method to remove bacterial biofilm from a stainless steel (SS) surface using a potential pulse/reverse pulse technique. This technique employs a periodic waveform that consists of anodic and cathodic pulses. The pulses can effectively strip a thin layer of metal off the SS surface, along with the adherent biofilm, in a saline solution. Not only can the pulses effectively remove biofilm from the SS surface, but they also regenerate the original mirror-like shiny surface. The importance of this electrochemical biofilm removal method is its wide applicability for any types of biofilms. That is, instead of directly removing the biofilm, it removes a very thin layer of the metal under the biofilm. Thus, the removal process is independent to the nature of the biofilms. Furthermore, this electrochemical biofilm removal method is rapid (less than 30?s of potential pulse time) and does not require hazardous chemicals.     

Quantification of calcium entry at the T-tubules and surface membrane in rat ventricular myocytes

The action potential of cardiac ventricular myocytes is characterized by its long duration, mainly due to Ca flux through L-type Ca channels. Ca entry also serves to trigger the release of Ca from the sarcoplasmic reticulum. The aim of this study was to investigate the role of cell membrane invaginations called transverse (T)-tubules in determining Ca influx and action potential duration in cardiac ventricular myocytes. We used the whole cell patch clamp technique to record electrophysiological activity in intact rat ventricular myocytes (i.e., from the T-tubules and surface sarcolemma) and in detubulated myocytes (i.e., from the surface sarcolemma only). Action potentials were significantly shorter in detubulated cells than in control cells. In contrast, resting membrane potential and action potential amplitude were similar in control and detubulated myocytes. Experiments under voltage clamp using action potential waveforms were used to quantify Ca entry via the Ca current. Ca entry after detubulation was reduced by approximately 60%, a value similar to the decrease in action potential duration. We calculated that Ca influx at the T-tubules is 1.3 times that at the cell surface (4.9 vs. 3.8 micromol/L cytosol, respectively) during a square voltage clamp pulse. In contrast, during a cardiac action potential, Ca entry at the T-tubules is 2.2 times that at the cell surface (3.0 vs. 1.4 micromol/L cytosol, respectively). However, more Ca entry occurs per microm(2) of junctional membrane at the cell surface than in the T-tubules (in nM/microm(2): 1.43 vs. 1.06 during a cardiac action potential). This difference is unlikely to be due to a difference in the number of Ca channels/junction at each site because we estimate that the same number of Ca channels is present at cell surface and T-tubule junctions ( approximately 35). This study provides the first evidence that the T-tubules are a key site for the regulation of action potential duration in ventricular cardiac myocytes. Our data also provide the first direct measurements of T-tubular Ca influx, which are consistent with the idea that cardiac excitation-contraction coupling largely occurs at the T-tubule dyadic clefts.     

Activity analysis of thermal imaging videos using a difference imaging approach

Infrared thermal imaging is a passive imaging technique that captures the emitted radiation from an object to estimate surface temperature, often for inference of heat transfer. Infrared thermal imaging offers the potential to detect movement without the challenges of glare, shadows, or changes in lighting associated with visual digital imaging or active infrared imaging. In this paper, we employ a frame subtraction algorithm for extracting the pixel-by-pixel relative change in signal from a fixed focus video file, tailored for use with thermal imaging videos. By summing the absolute differences across an entire video, we are able to assign quantitative activity assessments to thermal imaging data for comparison with simultaneous recordings of metabolic rates. We tested the accuracy and limits of this approach by analyzing movement of a metronome and provide an example application of the approach to a study of Darwin's finches. In principle, this “Difference Imaging Thermography” (DIT) would allow for activity data to be standardized to energetic measurements and could be applied to any radiometric imaging system.     

Uptake of Granulovirus from the Surface of Apples and Leaves by First Instar Larvae of the Codling Moth Cydia pomonella L. (Lepidoptera: Olethreutidae)

Cydia pomonella granulovirus (CpGV) has received considerable attention as a potential microbial insecticide for the control of the codling moth (Cydia pomonella L.) , a worldwide pest of apples. Laboratory experiments were established to investigate virus uptake by first instar larvae, using a novel leaf disc bioassay technique. Virus uptake was found to be independent of active feeding and larvae became infected simply by walking or browsing on sprayed leaf disc surfaces in as little time as 3.5 min. Infection increased as a function of time spent on the leaf disc surface and a linear log time/probit mortality relationship could be fitted. The bioassay technique used has potential for the realistic laboratory testing of virus spray formulations. A field experiment showed that virus infection could be contracted by newly hatched codling moth larvae both from the surface of sprayed leaves and sprayed fruit. The potential for exploiting this knowledge for improving spray formulations is discussed.     

Surface engineering of living myoblasts via selective periodate oxidation

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.     

Rapid and quantitative detection of the microbial spoilage of meat by fourier transform infrared spectroscopy and machine learning

Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable "fingerprints." Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial least-squares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10(7) bacteria.g(-1) the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.     

Low-level exertions of the neck musculature: a study of research methods.

Musculoskeletal neck discomfort is prevalent in many occupations and has been the focus of much research employing surface electromyography (sEMG). Significant differences in experimental methods among researchers make comparisons across studies difficult. The goal of the current research was to use empirical methods to answer specific methodological questions concerning use of sEMG in evaluation of the neck extensor system. This was accomplished in two studies. In Experiment 1, ultrasound technology was used to: (a) determine accessibility of m. splenius and semispinalis capitis with surface electrodes, (b) identify appropriate electrode locations for these muscles/muscle groups, and (c) illustrate potential benefits of using ultrasound in locating muscles/placing electrodes. Experiment 2 sought to assess effects of posture when normalizing sEMG data. Results from Experiment 1 showed no direct access to semispinalis capitis for surface electrodes; their activity can only be sampled as part of a group of muscles. In most subjects, m. splenius was found to be accessible to surface electrodes. Electrode placement recommendations are provided. Results of Experiment 2 showed significant differences in normalized EMG data between a posture-specific technique and a reference posture technique. Posture-specific normalization is recommended for accurately assessing the relative intensity of contractions of these muscles.     

A fiber optic biosensor (FOBS) to monitor mutans streptococci in human saliva

A fiber optic biosensor (FOBS) to monitor mutans streptococci activity in human saliva is developed. The biosensor utilizes e fiber optic evanescent wave spectroscopy to monitor a bacterial mediated biochemical reaction. To achieve this, a short length of the cladding is removed; the fiber core surface is treated and coated with a thin film of porous glass medium using sol-gel technique. The mutans streptococci mediated reaction with sucrose is monitored using a photosensitive indicator, which is immobilized within the porous glass coating. Spectroscopic analysis shows that the transmitted intensity at 597 nm increases conspicuously when monitored for 120 min. Two distinct phases are observed, one from 0 to 60 min and the other from 60 to 120 min. A negative correlation coefficient between the rate of increase in absorption peak intensity recorded by the FOBS and the decrease in pH measured using the pH meter, was calculated to be rho=-0.994. This investigation highlights the potential benefits of this sensor to monitor mutans streptococci activity in saliva.     

Rapid MALDI-TOF-MS analysis in the study of interaction between whole bacterial cells and human target molecules: binding of Bifidobacterium to human plasminogen

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.     

Use of surface EMG for studying motor unit recruitment during isometric linear force ramp.

The aim of this work was to demonstrate the rank order of motor unit (MU) recruitment by surface EMG based on a Laplacian detection technique and to document the MU features at their recruitment threshold. Surface EMG signals were recorded on the biceps brachii of 10 healthy subjects during linear force ramps. When achievable, the signals were decomposed into MU action potential (MUAP) trains. MU inter-pulse interval (IPI), conduction velocity (MUCV) and amplitude were estimated on the first 12 MUAPs of each detectable train in order to characterize the MU features at their firing onset. A strong correlation was found between MU recruitment threshold and IPI, MUCV, and amplitude, showing that the size principle can be demonstrated by a fully non-invasive EMG technique. However, signal decomposition was not possible on seven subjects due to the effects of the volume conductor when the skinfold thickness was too large. When requirements for an optimal detection of MUAP trains are met, surface EMG may be used to improve our understanding of MU activity.     

Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.