Vibrational spectroscopy techniques, such as Fourier-transform infrared (FTIR) and Raman spectroscopy, have been successful methods for studying the interaction of light with biological materials and ...facilitating novel cell biology analysis. Spectrochemical analysis is very attractive in disease screening and diagnosis, microbiological studies and forensic and environmental investigations because of its low cost, minimal sample preparation, non-destructive nature and substantially accurate results. However, there is now an urgent need for multivariate classification protocols allowing one to analyze biologically derived spectrochemical data to obtain accurate and reliable results. Multivariate classification comprises discriminant analysis and class-modeling techniques where multiple spectral variables are analyzed in conjunction to distinguish and assign unknown samples to pre-defined groups. The requirement for such protocols is demonstrated by the fact that applications of deep-learning algorithms of complex datasets are being increasingly recognized as critical for extracting important information and visualizing it in a readily interpretable form. Hereby, we have provided a tutorial for multivariate classification analysis of vibrational spectroscopy data (FTIR, Raman and near-IR) highlighting a series of critical steps, such as preprocessing, data selection, feature extraction, classification and model validation. This is an essential aspect toward the construction of a practical spectrochemical analysis model for biological analysis in real-world applications, where fast, accurate and reliable classification models are fundamental.
The cytochrome P450 monooxygenases (P450s) are thiolate heme proteins that can, often under physiological conditions, catalyze many distinct oxidative transformations on a wide variety of molecules, ...including relatively simple alkanes or fatty acids, as well as more complex compounds such as steroids and exogenous pollutants. They perform such impressive chemistry utilizing a sophisticated catalytic cycle that involves a series of consecutive chemical transformations of heme prosthetic group. Each of these steps provides a unique spectral signature that reflects changes in oxidation or spin states, deformation of the porphyrin ring or alteration of dioxygen moieties. For a long time, the focus of cytochrome P450 research was to understand the underlying reaction mechanism of each enzymatic step, with the biggest challenge being identification and characterization of the powerful oxidizing intermediates. Spectroscopic methods, such as electronic absorption (UV–Vis), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electron nuclear double resonance (ENDOR), Mössbauer, X-ray absorption (XAS), and resonance Raman (rR), have been useful tools in providing multifaceted and detailed mechanistic insights into the biophysics and biochemistry of these fascinating enzymes. The combination of spectroscopic techniques with novel approaches, such as cryoreduction and Nanodisc technology, allowed for generation, trapping and characterizing long sought transient intermediates, a task that has been difficult to achieve using other methods. Results obtained from the UV–Vis, rR and EPR spectroscopies are the main focus of this review, while the remaining spectroscopic techniques are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
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Vibrational spectroscopy (mid-infrared (IR) and Raman) and its fingerprinting capabilities offer rapid, high-throughput, and non-destructive analysis of a wide range of sample types producing a ...characteristic chemical "fingerprint" with a unique signature profile. Nuclear magnetic resonance (NMR) spectroscopy and an array of mass spectrometry (MS) techniques provide selectivity and specificity for screening metabolites, but demand costly instrumentation, complex sample pretreatment, are labor-intensive, require well-trained technicians to operate the instrumentation, and are less amenable for implementation in clinics. The potential for vibration spectroscopy techniques to be brought to the bedside gives hope for huge cost savings and potential revolutionary advances in diagnostics in the clinic. We discuss the utilization of current vibrational spectroscopy methodologies on biologic samples as an avenue towards rapid cost saving diagnostics.
Background
While the excisional biopsy and histological examination of suspicious lesions remains the current gold standard for diagnosing cutaneous melanoma (CM), there is a demand for more ...objective and non‐invasive examination methods that may support clinicians in their decision when to biopsy or not.
Methods
This review is based on publications and guidelines retrieved by a selective search in PubMed and MEDLINE and focused on non‐invasive diagnostic strategies for detecting melanoma.
Results
Ten different non‐invasive techniques were compared with regard to applicability, status of development, and resources necessary for introduction into clinical routine (dermoscopy, sequential digital dermoscopy, total body photography, computer‐aided multispectral digital analysis, electrical impedance spectroscopy, Raman spectroscopy, reflectance confocal microscopy, multiphoton tomography, stepwise two‐photon‐laser spectroscopy, quantitative dynamic infrared imaging). In an effort to create a classification based on our analyses, we suggest to differentiate i) tools for screening of patients in daily clinical routine, ii) tools for examination of a restricted number of preselected lesions that produce an automated diagnostic score, iii) tools for examination of a restricted number of preselected lesions at specialized centers requiring extensive training, iv) devices at an experimental stage of development.
Conclusion
None of the discussed examination techniques is able to provide a definite and final diagnosis or to completely replace the histopathological examination. Up to date, the need for fully automated devices offering a complete skin cancer screening has not been satisfied.
Stable isotopes are extensively used as tracers for the study of plant-water sources. Isotope-ratio infrared spectroscopy (IRIS) offers a cheaper alternative to isotope-ratio mass spectroscopy ...(IRMS), but its use in studying plant and soil water is limited by the spectral interference caused by organic contaminants. Here, we examine two approaches to cope with contaminated samples in IRIS: on-line oxidation of organic compounds (MCM) and post-processing correction.
We assessed these methods compared to IRMS across 136 samples of xylem and soil water, and a set of ethanol– and methanol–water mixtures.
A post-processing correction significantly improved IRIS accuracy in both natural samples and alcohol dilutions, being effective with concentrations up to 8% of ethanol and 0.4% of methanol. MCM outperformed the post-processing correction in removing methanol interference, but did not effectively remove interference for high concentrations of ethanol.
By using both approaches, IRIS can overcome with reasonable accuracy the analytical uncertainties associated with most organic contaminants found in soil and xylem water. We recommend the post-processing correction as the first choice for analysis of samples of unknown contamination. Nevertheless, MCM can be more effective for evaluating samples containing contaminants responsible for strong spectral interferences at low concentrations, such as methanol.
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