共查询到20条相似文献,搜索用时 0 毫秒
1.
Victor Yashunsky Leorah Kharilker Efrat Zlotkin-Rivkin David Rund Naomi Melamed-Book Eitan Erez Zahavi Eran Perlson Silvana Mercone Michael Golosovsky Dan Davidov Benjamin Aroeti 《PloS one》2013,8(10)
Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell''s height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity. 相似文献
2.
Fourier transform infrared (FTIR) spectroscopy has been established as a fast spectroscopic method for biochemical analysis of cells and tissues. In this research we aimed to investigate FTIR's utility for identifying and characterizing different modes of cell death, using leukemic cell lines as a model system. CCRF-CEM and U937 leukemia cells were treated with arabinoside and doxorubicin apoptosis inducers, as well as with potassium cyanide, saponin, freezing-thawing, and H2O2 necrosis inducers. Cell death mode was determined by various gold standard biochemical methods in parallel with FTIR-microscope measurements. Both cell death modes exhibit large spectral changes in lipid absorbance during apoptosis and necrosis; however, these changes are similar and thus cannot be used to distinguish apoptosis from necrosis. In contrast to the above confounding factor, our results reveal that apoptosis and necrosis can still be distinguished by the degree of DNA opaqueness to infrared light. Moreover, these two cell death modes also can be differentiated by their infrared absorbance, which relates to the secondary structure of total cellular protein. In light of these findings, we conclude that, because of its capacity to monitor multiple biomolecular parameters, FTIR spectroscopy enables unambiguous and easy analysis of cell death modes and may be useful for biochemical and medical applications. 相似文献
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Koji Kato Masami Nitta Takashi Mizuno 《Bioscience, biotechnology, and biochemistry》2013,77(2):433-435
Bacterial production of 4-hydroxy(3,4-d)pyrazolopyrirnidine riboside (AP–R) was studied. Seven among 73 tested strains were found to produce AP–R through N-ribosyl transfer reaction between uridine and 4-hydroxy(3,4-d)pyrazolopyrimidine (allopurinol). AP–R was produced by the cell-free extract of Erwinia carotovora and was isolated in crystals from the reaction mixture. The crystalline AP–R was characterized by spectroscopic data and was confirmed to be β-1-ribosyl allopurinol. AP–R could not substitute for inosine to support the growth of a nonexacting purine base-requiring mutant of Sarcina lutea. 相似文献
5.
Background
Fungal spores and plant pollen cause respiratory diseases in susceptible individuals, such as asthma, allergic rhinitis and hypersensitivity pneumonitis. Aeroallergen monitoring networks are an important part of treatment strategies, but unfortunately traditional analysis is time consuming and expensive. We have explored the use of infrared spectroscopy of pollen and spores for an inexpensive and rapid characterization of aeroallergens.Methodology
The study is based on measurement of spore and pollen samples by single reflectance attenuated total reflectance Fourier transform infrared spectroscopy (SR-ATR FTIR). The experimental set includes 71 spore (Basidiomycota) and 121 pollen (Pinales, Fagales and Poales) samples. Along with fresh basidiospores, the study has been conducted on the archived samples collected within the last 50 years.Results
The spectroscopic-based methodology enables clear spectral differentiation between pollen and spores, as well as the separation of confamiliar and congeneric species. In addition, the analysis of the scattering signals inherent in the infrared spectra indicates that the FTIR methodology offers indirect estimation of morphology of pollen and spores. The analysis of fresh and archived spores shows that chemical composition of spores is well preserved even after decades of storage, including the characteristic taxonomy-related signals. Therefore, biochemical analysis of fungal spores by FTIR could provide economical, reliable and timely methodologies for improving fungal taxonomy, as well as for fungal identification and monitoring. This proof of principle study shows the potential for using FTIR as a rapid tool in aeroallergen studies. In addition, the presented method is ready to be immediately implemented in biological and ecological studies for direct measurement of pollen and spores from flowers and sporocarps. 相似文献6.
K Martinez-Cruz A Sepulveda-Jauregui N Escobar-Orozco F Thalasso 《Journal of microbiological methods》2012,91(1):89-92
Methanogenic activity (MA) tests are commonly carried out to estimate the capability of anaerobic biomass to treat effluents, to evaluate anaerobic activity in bioreactors or natural ecosystems, or to quantify inhibitory effects on methanogenic activity. These activity tests are usually based on the measurement of the volume of biogas produced by volumetric, pressure increase or gas chromatography (GC) methods. In this study, we present an alternative method for non-invasive measurement of methane produced during activity tests in closed vials, based on Infrared Tunable Diode Laser Absorption Spectroscopy (MA-TDLAS). This new method was tested during model acetoclastic and hydrogenotrophic methanogenic activity tests and was compared to a more traditional method based on gas chromatography. From the results obtained, the CH(4) detection limit of the method was estimated to 60ppm and the minimum measurable methane production rate was estimated to 1.09(.)10(-3)mgl(-1)h(-1), which is below CH(4) production rate usually reported in both anaerobic reactors and natural ecosystems. Additionally to sensitivity, the method has several potential interests compared to more traditional methods among which short measurements time allowing the measurement of a large number of MA test vials, non-invasive measurements avoiding leakage or external interferences and similar cost to GC based methods. It is concluded that MA-TDLAS is a promising method that could be of interest not only in the field of anaerobic digestion but also, in the field of environmental ecology where CH(4) production rates are usually very low. 相似文献
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Victoria Astley Kimberly Reichel Rajind Mendis Daniel M. Mittleman 《Journal of visualized experiments : JoVE》2012,(66)
Refractive index (RI) sensing is a powerful noninvasive and label-free sensing technique for the identification, detection and monitoring of microfluidic samples with a wide range of possible sensor designs such as interferometers and resonators 1,2. Most of the existing RI sensing applications focus on biological materials in aqueous solutions in visible and IR frequencies, such as DNA hybridization and genome sequencing. At terahertz frequencies, applications include quality control, monitoring of industrial processes and sensing and detection applications involving nonpolar materials.Several potential designs for refractive index sensors in the terahertz regime exist, including photonic crystal waveguides 3, asymmetric split-ring resonators 4, and photonic band gap structures integrated into parallel-plate waveguides 5. Many of these designs are based on optical resonators such as rings or cavities. The resonant frequencies of these structures are dependent on the refractive index of the material in or around the resonator. By monitoring the shifts in resonant frequency the refractive index of a sample can be accurately measured and this in turn can be used to identify a material, monitor contamination or dilution, etc.The sensor design we use here is based on a simple parallel-plate waveguide 6,7. A rectangular groove machined into one face acts as a resonant cavity (Figures 1 and 2). When terahertz radiation is coupled into the waveguide and propagates in the lowest-order transverse-electric (TE1) mode, the result is a single strong resonant feature with a tunable resonant frequency that is dependent on the geometry of the groove 6,8. This groove can be filled with nonpolar liquid microfluidic samples which cause a shift in the observed resonant frequency that depends on the amount of liquid in the groove and its refractive index 9.Our technique has an advantage over other terahertz techniques in its simplicity, both in fabrication and implementation, since the procedure can be accomplished with standard laboratory equipment without the need for a clean room or any special fabrication or experimental techniques. It can also be easily expanded to multichannel operation by the incorporation of multiple grooves 10. In this video we will describe our complete experimental procedure, from the design of the sensor to the data analysis and determination of the sample refractive index. 相似文献
8.
Michael Kümmerle Siegfried Scherer Herbert Seiler 《Applied and environmental microbiology》1998,64(6):2207-2214
Computer-based Fourier-transform infrared spectroscopy (FT-IR) was used to identify food-borne, predominantly fermentative yeasts. Dried yeast suspensions provided the films suitable for FT-IR measurement. Informative windows in the spectrum were selected and combined to achieve optimal results. A reference spectrum library was assembled, based on 332 defined yeast strains from international yeast collections and our own isolates. All strains were identified with conventional methods using physiological and morphological characteristics. In order to assess identification quality, another 722 unknown yeast isolates not included in the reference spectrum library were identified both by classical methods and by comparison of their FT-IR spectra with those of the reference spectrum library. Ninety-seven and one-half percent of these isolates were identified correctly by FT-IR. Easy handling, rapid identification within 24 h when starting from a single colony, and a high differentiation capacity thus render FT-IR technology clearly superior to other routine methods for the identification of yeasts.Yeasts not only provided humans with the first biotechnologically produced food such as wine, bread, and fermented milk products but are also responsible for food spoilage (19), and some species are of medical importance. Therefore, a reliable method of yeast identification is economically significant (40). Furthermore, until now about 700 yeast species have been described. Since only a few habitats have been investigated in detail so far, a wide range of yeasts is likely to be discovered in the future (6). Exploration of new species includes the identification of a large number of isolates in order to eliminate duplicates and to discover unusual forms. For such tasks, a rapid, simple, low-cost identification method is needed. Conventional differentiation systems using morphological characters as well as patterns of the assimilation and fermentation of carbon sources (4, 22, 35) do not fulfil these requirements (9, 33, 38, 40). They are tedious and time-consuming, and, quite often, their capacity is limited since many species are distinguished from one another by a single physiological reaction which is often controlled by only one mutable marker (4, 20).Alternative methods such as fatty acid analysis (1, 31), electrophoretic karyotyping (10), restriction fragment length polymorphism, and DNA fingerprinting (26, 37) have already been evaluated (8). Restriction enzyme analysis of PCR-amplified rDNA (2), randomly amplified polymorphic DNA (3, 27), and nucleic acid hybridization with oligonucleotide probes (21, 24) have also been used. While some of these techniques do provide satisfactory results, molecular methods in general are still difficult to perform on a routine basis in laboratories of the food industry.Fourier-transform infrared (FT-IR) spectroscopy is used for the identification of substances in chemical analyses (14). The wavelength of infrared radiation ranges from 1 μm to 1 mm (32). In general, the wave number ν, the reciprocal of the wavelength, is used as a physical unit for FT-IR spectroscopy. Infrared radiation is divided into near (ν = 12,500 to 4,000 cm−1), middle (ν = 4,000 to 200 cm−1), and far (ν = 200 to 10 cm−1) infrared. In this work, only the middle infrared section was used. FT-IR spectroscopy involves the observation of vibrations of molecules that are excited by an infrared beam. Molecules are able to absorb the energy of distinct light quanta and start a rocking or rotation movement. The FT-IR spectrum uses only vibrations that lead to a change in the dipole moment (14). An infrared spectrum represents a fingerprint which is characteristic for any chemical substance.The composition of biological material and, thus, of its FT-IR spectrum, is exceedingly complex, representing a characteristic fingerprint. Some years ago, Naumann and coworkers suggested identifying microorganisms by FT-IR spectroscopy (28–30). In principle, a reference spectrum library is assembled based on well-characterized strains and species. The FT-IR spectrum of any unidentified isolate is then measured under the same conditions as those used for the reference spectra and is compared to spectra in the reference spectrum library. If the library contains an identical or a very similar spectrum, an identification is possible. The success of the method is, therefore, directly dependent on the complexity of the reference spectrum library. The application of FT-IR spectroscopy has been reported for some species of the genera Lactobacillus (7), Actinomyces (15), Listeria (18), Streptococcus (13), and Clostridium (11). There are two reports which present preliminary data indicating that eukaryotic microorganisms such as yeasts may also be identified by FT-IR (17, 36). However, all these studies are based on a very limited number of species and isolates. For verification of the method only a few strains, which often were part of the reference spectrum library as well, were used. It was, therefore, still unclear whether FT-IR spectroscopy indeed was a competitive identification method.The aim of this study was to develop a standardized sample preparation procedure for yeasts (suitable for the normal laboratory), to select the most significant spectral windows for efficient identification, and to assemble a spectral reference library of sufficient complexity. Last, the identification of a great variety of unknown yeast isolates by FT-IR spectroscopy and conventional techniques had to be done in order to verify the method. 相似文献
9.
Achim Kohler Ulrike B?cker Volha Shapaval Annabelle Forsmark Mats Andersson Jonas Warringer Harald Martens Stig W. Omholt Anders Blomberg 《PloS one》2015,10(2)
Single-channel optical density measurements of population growth are the dominant large scale phenotyping methodology for bridging the gene-function gap in yeast. However, a substantial amount of the genetic variation induced by single allele, single gene or double gene knock-out technologies fail to manifest in detectable growth phenotypes under conditions readily testable in the laboratory. Thus, new high-throughput phenotyping technologies capable of providing information about molecular level consequences of genetic variation are sorely needed. Here we report a protocol for high-throughput Fourier transform infrared spectroscopy (FTIR) measuring biochemical fingerprints of yeast strains. It includes high-throughput cultivation for FTIR spectroscopy, FTIR measurements and spectral pre-treatment to increase measurement accuracy. We demonstrate its capacity to distinguish not only yeast genera, species and populations, but also strains that differ only by a single gene, its excellent signal-to-noise ratio and its relative robustness to measurement bias. Finally, we illustrated its applicability by determining the FTIR signatures of all viable Saccharomyces cerevisiae single gene knock-outs corresponding to lipid biosynthesis genes. Many of the examined knock-out strains showed distinct, highly reproducible FTIR phenotypes despite having no detectable growth phenotype. These phenotypes were confirmed by conventional lipid analysis and could be linked to specific changes in lipid composition. We conclude that the introduced protocol is robust to noise and bias, possible to apply on a very large scale, and capable of generating biologically meaningful biochemical fingerprints that are strain specific, even when strains lack detectable growth phenotypes. Thus, it has a substantial potential for application in the molecular functionalization of the yeast genome. 相似文献
10.
Saroj Kumar 《Biophysical journal》2010,98(9):1931-1940
Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-13C2-labeled PEP. We obtained the following assignments: 1589 cm−1 (antisymmetric carboxylate stretching vibration); 1415 cm−1 (symmetric carboxylate stretching vibration); 1214 cm−1 (C-O stretching vibration); 1124 and 1110 cm−1 (asymmetric PO32- stretching vibrations); and 967 cm−1 (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm−1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases. 相似文献
11.
《Biophysical journal》2020,118(11):2694-2702
Calcium ions bind to lipid membranes containing anionic lipids; however, characterizing the specific ion-lipid interactions in multicomponent membranes has remained challenging because it requires nonperturbative lipid-specific probes. Here, using a combination of isotope-edited infrared spectroscopy and molecular dynamics simulations, we characterize the effects of a physiologically relevant (2 mM) Ca2+ concentration on zwitterionic phosphatidylcholine and anionic phosphatidylserine lipids in mixed lipid membranes. We show that Ca2+ alters hydrogen bonding between water and lipid headgroups by forming a coordination complex involving the lipid headgroups and water. These interactions distort interfacial water orientations and prevent hydrogen bonding with lipid ester carbonyls. We demonstrate, experimentally, that these effects are more pronounced for the anionic phosphatidylserine lipids than for zwitterionic phosphatidylcholine lipids in the same membrane. 相似文献
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氨基酸酯化方法的比较及其红外光谱研究 总被引:7,自引:0,他引:7
在合成氨基酸酯锗类化合物时,制备了八种氨基酸酯,对文献提供的六种方法进行了实验比较,选出经济简便、产率高的方法。并对氨基酸及其酯的红外光谱的变化特征进行了研究。 相似文献
14.
红外光谱技术在生物过程监测中的应用 总被引:5,自引:0,他引:5
在线监测化学组分的浓度对许多生物过程都是十分必要的。然而,探头需耐高温灭菌的要求和生物体系自身的复杂性给许多分析技术的在线监测带来了困难。近几年,随仪器和数据处理技术的迅速发展,应用红外光谱技术对生物过程的原位或在线监测日益广泛。本文对红外过程分析技术进行了较全面的综述,介绍了红外分析的原理、进展及在生物过程监测中的应用。 相似文献
15.
Günnur Güler Rebecca M. G?rtner Christine Ziegler Werner M?ntele 《The Journal of biological chemistry》2016,291(9):4295-4307
The Na+-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K+ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K+ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions. 相似文献
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Samy Nagib J?rg Rau Osama Sammra Christoph L?mmler Karen Schlez Michael Zsch?ck Ellen Prenger-Berninghoff Guenter Klein Amir Abdulmawjood 《PloS one》2014,9(8)
The present study was designed to investigate the potential of Fourier transform infrared (FT-IR) spectroscopy to identify Trueperella (T.) pyogenes isolated from bovine clinical mastitis. FT-IR spectroscopy was applied to 57 isolates obtained from 55 cows in a period from 2009 to 2012. Prior to FT-IR spectroscopy these isolates were identified by phenotypic and genotypic properties, also including the determination of seven potential virulence factor encoding genes. The FT-IR analysis revealed a reliable identification of all 57 isolates as T. pyogenes and a clear separation of this species from the other species of genus Trueperella and from species of genus Arcanobacterium and Actinomyces. The results showed that all 57 isolates were assigned to the correct species indicating that FT-IR spectroscopy could also be efficiently used for identification of this bacterial pathogen. 相似文献
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Sean T. Vittadello Scott W. McCue Gency Gunasingh Nikolas K. Haass Matthew J. Simpson 《Biophysical journal》2018,114(5):1241-1253
The fluorescent ubiquitination-based cell cycle indicator, also known as FUCCI, allows the visualization of the G1 and S/G2/M cell cycle phases of individual cells. FUCCI consists of two fluorescent probes, so that cells in the G1 phase fluoresce red and cells in the S/G2/M phase fluoresce green. FUCCI reveals real-time information about cell cycle dynamics of individual cells, and can be used to explore how the cell cycle relates to the location of individual cells, local cell density, and different cellular microenvironments. In particular, FUCCI is used in experimental studies examining cell migration, such as malignant invasion and wound healing. Here we present, to our knowledge, new mathematical models that can describe cell migration and cell cycle dynamics as indicated by FUCCI. The fundamental model describes the two cell cycle phases, G1 and S/G2/M, which FUCCI directly labels. The extended model includes a third phase, early S, which FUCCI indirectly labels. We present experimental data from scratch assays using FUCCI-transduced melanoma cells, and show that the predictions of spatial and temporal patterns of cell density in the experiments can be described by the fundamental model. We obtain numerical solutions of both the fundamental and extended models, which can take the form of traveling waves. These solutions are mathematically interesting because they are a combination of moving wavefronts and moving pulses. We derive and confirm a simple analytical expression for the minimum wave speed, as well as exploring how the wave speed depends on the spatial decay rate of the initial condition. 相似文献
20.
Jeremy?S. Treger Michael?F. Priest Raymond Iezzi Francisco Bezanilla 《Biophysical journal》2014,107(6):L09-L12
Clinical methods used to assess the electrical activity of excitable cells are often limited by their poor spatial resolution or their invasiveness. One promising solution to this problem is to optically measure membrane potential using a voltage-sensitive dye, but thus far, none of these dyes have been available for human use. Here we report that indocyanine green (ICG), an infrared fluorescent dye with FDA approval as an intravenously administered contrast agent, is voltage-sensitive. The fluorescence of ICG can follow action potentials in artificial neurons and cultured rat neurons and cardiomyocytes. ICG also visualized electrical activity induced in living explants of rat brain. In humans, ICG labels excitable cells and is routinely visualized transdermally with high spatial resolution. As an infrared voltage-sensitive dye with a low toxicity profile that can be readily imaged in deep tissues, ICG may have significant utility for clinical and basic research applications previously intractable for potentiometric dyes.Voltage-sensitive dyes provide a way to observe cellular electrical activity without the physical limitations imposed by electrodes. Although these dyes can monitor membrane potential with a resolution of a few microns from large populations of cells (1), there are three obstacles that prevent the use of these dyes in many research settings, including clinical research:
- 1.Most voltage-sensitive dyes use visible wavelengths of light that prevent imaging of tissues beneath the skin.
- 2.Many of these dyes produce significant toxicity or off-target effects (2).
- 3.Before this report, to our knowledge, no voltage-sensitive dyes have ever been available for administration in humans, which has limited their value in biomedically focused research.