Dataset Information

Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy.

ABSTRACT: The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue.

SUBMITTER: Whelan DR

PROVIDER: S-EPMC3141270 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy.

Whelan Donna R DR Bambery Keith R KR Heraud Philip P Tobin Mark J MJ Diem Max M McNaughton Don D Wood Bayden R BR

Nucleic acids research 20110329 13

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-li ...[more]

PMID: 21447564

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Project description:Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy.

Project description:Despite the increasing popularity of liquid chromatography−mass spectrometry (LC-MS)-based lipidomics, there is a lack of accepted and validated methods for lipid extract quality and quantity assessment prior to LC-MS. Fourier-Transform Infrared Spectroscopy (FTIR) has been reported for quantification of pure lipids. However, the impact of complex lipid sample complexity and purity on total lipid quantification accuracy has not been investigated. Here, we report comprehensive assessment of the sample matrix on the accuracy of lipid quantification using Attenuated Total Reflectance (ATR)-FTIR and establish a simple workflow for lipidomics sample quantification. We show that both pure and complex lipids show characteristic FTIR vibrations of CH- and C=O-stretching vibrations, with a quantitative range of 40−3000 ng and a limit of detection of 12 ng, but sample extraction method and local baseline subtraction during FTIR spectral processing significantly impact lipid quantification via CH stretching. To facilitate sample quality screening, we developed the Lipid Quality (LiQ) score from a spectral library of common contaminants, using a ratio of peak heights between CH stretching vibrations maxima and the collective vibrations from amide/amine, CH-stretching minima and sugar moieties. Taking all tested parameters together, we propose a rapid FTIR workflow for routine lipidomics sample quality and quantity assessment and tested this workflow by comparing to the total LC-MS intensity of targeted lipidomics of 107 human plasma lipid extracts. Exclusion of poor-quality samples based on LiQ score improved the correlation between FTIR and LC-MS quantification. The uncertainty of absolute quantification by FTIR was estimated using a 795 ng SPLASH LipidoMix standard to be <10%. With low sample requirement, we anticipate this simple and rapid method will enhance lipidomics workflow by enabling accurate total lipid quantification and normalization of lipid quantity for MS analysis.

Dataset Information

Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy.

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Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy.

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