Mitochondrial Membrane Studies Using Impedance Spectroscopy with Parallel pH Monitoring

A biological microelectromechanical system (BioMEMS) device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy) to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1) electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2) ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample’s complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.     

Investigation of the Variability of NIR In-line Monitoring of Roller Compaction Process by Using Fast Fourier Transform (FFT) Analysis

The purpose of this research was to investigate the variability of the roller compaction process while monitoring in-line with near-infrared (NIR) spectroscopy. In this paper, a pragmatic method in determining this variability of in-line NIR monitoring roller compaction process was developed and the variability limits were established. Fast Fourier Transform (FFT) analysis was used to study the source of the systematic fluctuations of the NIR spectra. An off-line variability analysis method was developed as well to simulate the in-line monitoring process in order to determine the variability limits of the roller compaction process. For this study, a binary formulation was prepared composed of acetaminophen and microcrystalline cellulose. Different roller compaction parameters such as roll speed and feeding rates were investigated to understand the variability of the process. The best-fit line slope of NIR spectra exhibited frequency dependence only on the roll speed regardless of the feeding rates. The eccentricity of the rolling motion of rollers was identified as the major source of variability and correlated with the fluctuations of the slopes of NIR spectra. The off-line static and dynamic analyses of the compacts defined two different variability of the roller compaction; the variability limits were established. These findings were proved critical in the optimization of the experimental setup of the roller compaction process by minimizing the variability of NIR in-line monitoring.     

Critical Evaluation of Product Ion Selection and Spectral Correlation Analysis for Biomarker Screening Using Targeted Peptide Multiple Reaction Monitoring

Introduction  Tandem mass spectrometry (MS/MS) has emerged as a cornerstone of proteomic screens aimed at discovering putative protein biomarkers of disease with potential clinical applications. Systematic validation of lead candidates in large numbers of samples from patient cohorts remains an important challenge. One particularly promising high throughout technique is multiple reaction monitoring (MRM), a targeted form of MS/MS by which precise peptide precursor–product ion combinations, or transitions, are selectively tracked as informative probes. Despite recent progress, however, many important computational and statistical issues remain unresolved. These include the selection of an optimal set of transitions so as to achieve sufficiently high specificity and sensitivity when profiling complex biological specimens, and the corresponding generation of a suitable scoring function to reliably confirm tentative molecular identities based on noisy spectra. Methods  In this study, we investigate various empirical criteria that are helpful to consider when developing and interpreting MRM-style assays based on the similarity between experimental and annotated reference spectra. We also rigorously evaluate and compare the performance of conventional spectral similarity measures, based on only a few pre-selected representative transitions, with a generic scoring metric, termed T _corr, wherein a selected product ion profile is used to score spectral comparisons. Conclusions  Our analyses demonstrate that T _corr is potentially more suitable and effective for detecting biomarkers in complex biological mixtures than more traditional spectral library searches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jian Liu and Johannes A Hewel contributed equally to this study.     

Determination of Abscisic Acid in Pinus densiflora by Selected Ion Monitoring

Abscisic acid (ABA) in the stem of akamatsu (Pinus densiflora) was identified and quantified by gas chromatography-mass spectrometry-selected ion monitoring using hexadeuterated ABA as an internal standard. tert-Butyldimethylsilyl ester was used as a derivative of ABA. This derivative had high sensitivity and selectivity for ABA determination. ABA concentrations in cambial region scrapings were independent of the cessation of cambial activity.     

Impedance Analysis of Ion Transport Through Supported Lipid Membranes Doped with Ionophores: A New Kinetic Approach

Kinetics of facilitated ion transport through planar bilayer membranes are normally analyzed by electrical conductance methods. The additional use of electrical relaxation techniques, such as voltage jump, is necessary to evaluate individual rate constants. Although electrochemical impedance spectroscopy is recognized as the most powerful of the available electric relaxation techniques, it has rarely been used in connection with these kinetic studies. According to the new approach presented in this work, three steps were followed. First, a kinetic model was proposed that has the distinct quality of being general, i.e., it properly describes both carrier and channel mechanisms of ion transport. Second, the state equations for steady-state and for impedance experiments were derived, exhibiting the input–output representation pertaining to the model’s structure. With the application of a method based on the similarity transformation approach, it was possible to check that the proposed mechanism is distinguishable, i.e., no other model with a different structure exhibits the same input–output behavior for any input as the original. Additionally, the method allowed us to check whether the proposed model is globally identifiable (i.e., whether there is a single set of fit parameters for the model) when analyzed in terms of its impedance response. Thus, our model does not represent a theoretical interpretation of the experimental impedance but rather constitutes the prerequisite to select this type of experiment in order to obtain optimal kinetic identification of the system. Finally, impedance measurements were performed and the results were fitted to the proposed theoretical model in order to obtain the kinetic parameters of the system. The successful application of this approach is exemplified with results obtained for valinomycin–K⁺ in lipid bilayers supported onto gold substrates, i.e., an arrangement capable of emulating biological membranes.     

Analysis of Root Growth by Impedance Spectroscopy (EIS)

Electrical impedance spectroscopy (EIS) is investigated as a non-destructive method for monitoring root growth of tomato. This paper aims to (i) review the basic principles of EIS applied to the characterisation of the different parts of the soil–root–stem-electrode continuum, (ii) experiment the validity of the relationship between root weight and root capacitance taking into account the influence of the soil and plant electrodes position, (iii) describe an EIS analysis of the root growth of tomato plants. All experiments were carried out in 50 dm³ containers either in hydroponics at 930 μS for the test of root fresh or dry weight and root capacitance relationships, or in a potting mix (oxisol) for electrode placement tests and EIS estimation of root growth. Electrical measurements of the soil–root–stem-electrode continuum were done with a two-electrode measuring system using unpolarisable Ag–AgCl electrodes. A ‘root cutting’ and a ‘progressively immersed root system’ experiments were carried out in order to validate the relationship between root capacitance and root mass at 1 kHz. The effects of soil electrode and plant electrode placement were also tested, pointing out the sensitivity of the method to the insertion height of the “plant electrode” into the stem. For the root growth experiment, Impedance Spectra (IS) measurements were made just before harvesting the roots for dry weight and length determination. Measurements were made 14, 22, 26 and 39 days after planting, until flowering. The IS of the soil–root–stem-electrode continuum was modelled by a lumped electric circuit consisting of a series resistor R ₀ for the soil and of four parallel resistance (R _i )-capacitance (C _i ) circuits for the other components of the circuit. The model had nine parameters whose values were estimated by Complex Nonlinear Least Squares curve fitting. A stepwise ascendant regression was used to identify the electrical parameters that better correlated with root dry mass or length increment: C ₃ and C ₄ were well correlated with root dry mass with a r ² of 0.975, whereas root length was explained by the combination of 1/R ₃, C ₃, 1/R ₂ and 1/R ₁ with a r ² of 0.986. This work may be considered as a new methodological contribution to the understanding of root electrical properties in the non-destructive diagnosis of root systems.     

Exhaled Breath Analysis for Lung Cancer Detection Using Ion Mobility Spectrometry

Background

Conventional methods for lung cancer detection including computed tomography (CT) and bronchoscopy are expensive and invasive. Thus, there is still a need for an optimal lung cancer detection technique.

Methods

The exhaled breath of 50 patients with lung cancer histologically proven by bronchoscopic biopsy samples (32 adenocarcinomas, 10 squamous cell carcinomas, 8 small cell carcinomas), were analyzed using ion mobility spectrometry (IMS) and compared with 39 healthy volunteers. As a secondary assessment, we compared adenocarcinoma patients with and without epidermal growth factor receptor (EGFR) mutation.

Results

A decision tree algorithm could separate patients with lung cancer including adenocarcinoma, squamous cell carcinoma and small cell carcinoma. One hundred-fifteen separated volatile organic compound (VOC) peaks were analyzed. Peak-2 noted as n-Dodecane using the IMS database was able to separate values with a sensitivity of 70.0% and a specificity of 89.7%. Incorporating a decision tree algorithm starting with n-Dodecane, a sensitivity of 76% and specificity of 100% was achieved. Comparing VOC peaks between adenocarcinoma and healthy subjects, n-Dodecane was able to separate values with a sensitivity of 81.3% and a specificity of 89.7%. Fourteen patients positive for EGFR mutation displayed a significantly higher n-Dodecane than for the 14 patients negative for EGFR (p<0.01), with a sensitivity of 85.7% and a specificity of 78.6%.

Conclusion

In this prospective study, VOC peak patterns using a decision tree algorithm were useful in the detection of lung cancer. Moreover, n-Dodecane analysis from adenocarcinoma patients might be useful to discriminate the EGFR mutation.     

Genetic Mutation Analysis of Human Gastric Adenocarcinomas Using Ion Torrent Sequencing Platform

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.     

In-Line Monitoring of a High-Shear Granulation Process Using the Baseline Shift of Near Infrared Spectra

Although near infrared (NIR) spectra are primarily influenced by undesired variations, i.e., baseline shifts and non-linearity, and many applications of NIR spectroscopy to the real-time monitoring of wet granulation processes have been reported, the granulation mechanisms behind these variations have not been fully discussed. These variations of NIR spectra can be canceled out using appropriate pre-processing techniques prior to spectral analysis. The present study assessed the feasibility of directly using baseline shifts in NIR spectra to monitor granulation processes, because such shifts can reflect changes in the physical properties of the granular material, including particle size, shape, density, and refractive index. Specifically, OPUSGRAN^®, a novel granulation technology, was investigated by in-line NIR monitoring. NIR spectra were collected using a NIR diffuse reflectance fiber optic probe immersed in a high-shear granulator while simultaneously examining the morphology, particle size, density, strength, and Raman images of the mixture during granulation. The NIR baseline shift pattern was found to be characteristic of the OPUSGRAN^® technology and was attributed to variations in the light transmittance, reflection, and scattering resulting from changes in the physicochemical properties of the samples during granulation. The baseline shift also exhibited an inflection point around the completion of granulation, and therefore may be used to determine the endpoint of the process. These results suggest that a specific pattern of NIR baseline shifts are associated with the unique OPUSGRAN^® granulation mechanism and can be applied to monitor the manufacturing process and determine the endpoint.     

Monitoring Urban Greenness Dynamics Using Multiple Endmember Spectral Mixture Analysis

Urban greenness is increasingly recognized as an essential constituent of the urban environment and can provide a range of services and enhance residents’ quality of life. Understanding the pattern of urban greenness and exploring its spatiotemporal dynamics would contribute valuable information for urban planning. In this paper, we investigated the pattern of urban greenness in Hangzhou, China, over the past two decades using time series Landsat-5 TM data obtained in 1990, 2002, and 2010. Multiple endmember spectral mixture analysis was used to derive vegetation cover fractions at the subpixel level. An RGB-vegetation fraction model, change intensity analysis and the concentric technique were integrated to reveal the detailed, spatial characteristics and the overall pattern of change in the vegetation cover fraction. Our results demonstrated the ability of multiple endmember spectral mixture analysis to accurately model the vegetation cover fraction in pixels despite the complex spectral confusion of different land cover types. The integration of multiple techniques revealed various changing patterns in urban greenness in this region. The overall vegetation cover has exhibited a drastic decrease over the past two decades, while no significant change occurred in the scenic spots that were studied. Meanwhile, a remarkable recovery of greenness was observed in the existing urban area. The increasing coverage of small green patches has played a vital role in the recovery of urban greenness. These changing patterns were more obvious during the period from 2002 to 2010 than from 1990 to 2002, and they revealed the combined effects of rapid urbanization and greening policies. This work demonstrates the usefulness of time series of vegetation cover fractions for conducting accurate and in-depth studies of the long-term trajectories of urban greenness to obtain meaningful information for sustainable urban development.     

Reduction of Copper Ion Phytotoxicity Using Rhodococcus-Biosurfactants

Biology Bulletin - Abstract—The effect of copper on the germination of seeds of several agricultural crops, such as common vetch (Vicia saliva L.), white mustard (Sinapis alba L.), and...     

Enhanced Sensitivity for Selected Reaction Monitoring Mass Spectrometry-based Targeted Proteomics Using a Dual Stage Electrodynamic Ion Funnel Interface

Selected reaction monitoring mass spectrometry (SRM-MS) is playing an increasing role in quantitative proteomics and biomarker discovery studies as a method for high throughput candidate quantification and verification. Although SRM-MS offers advantages in sensitivity and quantification compared with other MS-based techniques, current SRM technologies are still challenged by detection and quantification of low abundance proteins (e.g. present at ∼10 ng/ml or lower levels in blood plasma). Here we report enhanced detection sensitivity and reproducibility for SRM-based targeted proteomics by coupling a nanospray ionization multicapillary inlet/dual electrodynamic ion funnel interface to a commercial triple quadrupole mass spectrometer. Because of the increased efficiency in ion transmission, significant enhancements in overall signal intensities and improved limits of detection were observed with the new interface compared with the original interface for SRM measurements of tryptic peptides from proteins spiked into non-depleted mouse plasma over a range of concentrations. Overall, average SRM peak intensities were increased by ∼70-fold. The average level of detection for peptides also improved by ∼10-fold with notably improved reproducibility of peptide measurements as indicated by the reduced coefficients of variance. The ability to detect proteins ranging from 40 to 80 ng/ml within mouse plasma was demonstrated for all spiked proteins without the application of front-end immunoaffinity depletion and fractionation. This significant improvement in detection sensitivity for low abundance proteins in complex matrices is expected to enhance a broad range of SRM-MS applications including targeted protein and metabolite validation.Although mass spectrometry (MS)-based proteomics is a promising high throughput technology for biomarker discovery and validation (1–5), only a handful of cancer biomarkers have been approved by the United States Food and Drug Administration for clinical use in the last decade (6, 7). Assuming that low abundance biomarkers do exist in the biofluids to be studied, the success of biomarker discovery efforts primarily depends on the sensitivity, accuracy, and robustness of the measurement technologies; the quality and size of patient cohorts and clinical samples and execution within the context of an overall difficult and expensive path to clinical application that encompasses discovery, verification, and validation stages (1, 5, 8–10). A multiplexed assay platform increasingly considered for biomarker verification is selected reaction monitoring (SRM)¹ by tandem mass spectrometry using e.g. a triple quadrupole (QqQ) mass spectrometer to attain high throughput quantitative measurements of targeted proteins in complex matrices (1, 11, 12).SRM utilizes two stages of mass filtering by selecting a specific analyte ion of interest (precursor ion) in the first stage followed by a specific fragment ion derived from the precursor (fragment ion) filter in the second stage after collision-activated dissociation. Typically, several transitions (precursor/fragment ion pairs) are monitored for greater selectivity and confidence in a targeted peptide assay, and large numbers of peptides can be monitored during a single LC-MS/MS analysis. The two-stage mass selection by individual quadrupoles enables more rapid and continuous monitoring of specific ions derived from analytes of interest such as peptides and leads to significantly enhanced detection sensitivity and quantitative accuracy compared with broad (i.e. non-targeted) LC-MS or LC-MS/MS measurements (11, 12). Both the sensitivity and selectivity of SRM-MS make this technique well suited for the targeted detection and quantification of low abundance proteins in highly complex biofluids (13–16). The precision and reproducibility of SRM-based measurements of proteins in plasma across different laboratories have recently been assessed (17).Despite its promise, present SRM measurements still do not provide sufficient sensitivity for reliable detection and quantification of low abundance proteins in biofluids (e.g. present in plasma at ∼10 ng/ml or lower levels) primarily because of factors related to high sample complexity and the large dynamic range of relative protein abundances (7, 18, 19). Given sufficient selectivity, the sensitivity achievable is generally related to the peptide MS and MS/MS signal intensities obtained. One of the key factors limiting peptide MS intensities is the significant ion losses encountered between the electrospray ionization (ESI) source and the interface to the mass spectrometer. In typical LC-ESI-MS interfaces, the mass spectrometer inlet (e.g. heated capillary followed by a skimmer) presently provides total ion utilization and ion transmission efficiencies on the order of ∼1% (20) due to a combination of limited ion sampling from the atmospheric pressure ion source into the inlet and inefficient transmission of ions entering the first reduced pressure stage of the mass spectrometer.The electrodynamic ion funnel (21), which has been developed to efficiently capture, focus, and transmit ions to the high vacuum region of the mass spectrometer, is expected to provide a large benefit to SRM analyses. The original ion funnel interfaces, which operated at a maximum of ∼5 torr, were able to enhance signal intensities for a variety of MS analyzers (22–24) by replacing the inefficient skimmer interface. Although achieving near lossless ion transmission to high vacuum, losses at the atmospheric pressure interface went unmitigated. More recently, a high pressure ion funnel interface capable of operating at a pressure of ∼30 torr was introduced (25). The higher operating pressures accommodated greater gas loads and enabled more efficient ion sampling from atmospheric pressure through a multicapillary inlet. With a dual ion funnel interface comprising a high pressure ion funnel with a heated multicapillary inlet followed by a standard ion funnel operated at 1–2 torrs, highly efficient ion sampling from atmospheric pressure to high vacuum is readily achieved.In this study, we report the enhanced sensitivity and reproducibility of SRM-based targeted proteomics measurements achieved by implementing a dual stage electrodynamic ion funnel interface that incorporates a multicapillary inlet with a triple quadrupole mass spectrometer. A series of LC-SRM-MS measurements were made using mouse plasma samples spiked with various concentrations of tryptic peptides from five standard proteins to evaluate the improvements in detection sensitivity and reproducibility attained by this modified interface relative to a standard Thermo (single capillary inlet/skimmer) interface. A ∼10-fold improvement in the limit of detection (LOD) as well as improved measurement reproducibility was achieved.     

Efficient Driving of Piezoelectric Transducers Using a Biaxial Driving Technique

Efficient driving of piezoelectric materials is desirable when operating transducers for biomedical applications such as high intensity focused ultrasound (HIFU) or ultrasound imaging. More efficient operation reduces the electric power required to produce the desired bioeffect or contrast. Our preliminary work [Cole et al. Journal of Physics: Condensed Matter. 2014;26(13):135901.] suggested that driving transducers by applying orthogonal electric fields can significantly reduce the coercivity that opposes ferroelectric switching. We present here the experimental validation of this biaxial driving technique using piezoelectric ceramics typically used in HIFU. A set of narrow-band transducers was fabricated with two sets of electrodes placed in an orthogonal configuration (following the propagation and the lateral mode). The geometry of the ceramic was chosen to have a resonance frequency similar for the propagation and the lateral mode. The average (± s.d.) resonance frequency of the samples was 465.1 (± 1.5) kHz. Experiments were conducted in which each pair of electrodes was driven independently and measurements of effective acoustic power were obtained using the radiation force method. The efficiency (acoustic/electric power) of the biaxial driving method was compared to the results obtained when driving the ceramic using electrodes placed only in the pole direction. Our results indicate that the biaxial method increases efficiency from 50% to 125% relative to the using a single electric field.     

A Simple Determination Method for Herbicides,NIP and CNP in Soil Using Single Ion Monitoring Mass Spectrometry

A purified extracellular endo β-1,3-xylanase (EC 3.2.1.32) from an isolated strain, Aspergillus terreus A-07, was found to hydrolyze 1,3-xylosyl linkages only. When rhodymenan (β-1,4 and β-1.3-linked xylan) was hydrolyzed by β-1,3-xylanase (EF-6), four β-1,4-linked xylooligosaccharide fractions were produced. The main product was β-1,4-xylotriose, with trace amounts of other β-1,4-linked xylooligosaccharides. Successive degradation by β-l,4-xylosidase of the β,4-xylooligosaccharides that were produced from hydrolysis of β-1,3-xylanase on rhodymenan yielded only xylose as the final product.

We compared the action pattern of this enzyme with that of an extracellular endo β-l,4-xylanase (EC 3.2.1.8) of Streptomyces. From a mixture of products of β-1,4-xylanase hydrolysis on rhodymenan, an isomeric xylotriose was isolated by charcoal chromatography after treating with β-1.4-xylosidase. The structure of this isomeric xylotriose was elucidated by methylation analysis and its susceptibility to β-1,4-xylanase, β-1,3-xylanase, and β-1,4-xylosidase. The obtained isomeric xylotriose was identified as 3-O-β-xylopyranosyl-4-O-β-D-xylopyranosyl-D-xylose (X1→3X1→4X). It has a melting point of 224~225°C and ${\left[\alpha \right]}_{\text{D}}^{20}$(c = 1, H₂O)= —46°.     

Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

Recovery of Nuclease Produced by Lactococcus Lactis Using Expanded Bed Ion Exchange Chromatography

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml⁻¹. The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield.     

Measurements of Photosynthetic Sulfide Oxidation by Chlorobium Using a Sulfide Ion Selective Electrode

A simple technique is described for using a sulfide sensitiveelectrode to measure the photooxidation of H₂S by a green sulfurbacterium, Chlorobium limicola forma thiosulfatophilum. Sulfidephotooxidation occurred only in the presence of bicarbonateat concentrations greater than 0.1 mM. This implies that therate-limiting carboxylating enzyme for CO₂ fixation in Chlorobiumhas a relatively low affinity for CO₂ compared to ribulose-1,5-biphosphatecarboxylase. Carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone(FCCP), an uncoupler of photophosphorylation, delays sulfideoxidation for about 15 sec after the onset of illumination at2 µM and is completely inhibitory at 10 µM. Theseeffects can be explained by the ATP requirement for CO₂ fixation.When the photooxidation of H₂S was prevented by 10 µMFCCP, a photoevolution of H₂S was observed. (Received December 24, 1981; Accepted September 10, 1982)     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.