scholarly journals Harnessing intrinsic fluorescence for typing of secondary structures of DNA

2020 ◽  
Vol 48 (11) ◽  
pp. e61-e61 ◽  
Author(s):  
Michela Zuffo ◽  
Aurélie Gandolfini ◽  
Brahim Heddi ◽  
Anton Granzhan
Keyword(s):  
Nucleic Acids ◽  
High Throughput ◽  
Conformational Changes ◽  
Emission Spectra ◽  
Principal Component ◽  
Structural Diversity ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Linear Discriminant ◽  
Effective Manner

Abstract High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T, the most fluorescent G4 structure reported to date).

scholarly journals Harnessing intrinsic fluorescence for typing of secondary structures of DNA

2020 ◽  
Author(s):  
Michela Zuffo ◽  
Aurélie Gandolfini ◽  
Brahim Heddi ◽  
Anton Granzhan
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
Emission Spectra ◽  
Principal Component ◽  
Secondary Structures ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Linear Discriminant ◽  
Effective Manner

ABSTRACTDNA is polymorphic since, despite its ubiquitous presence as a double-stranded helix, it is able to fold into a plethora of other secondary structures both in vitro and in cells. Despite the considerable advances that have been made in understanding this structural diversity, its high-throughput investigation still faces severe limitations. This mainly stems from the lack of suitable label-free methods, combining a fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of the suitability of this phenomenon for tracking the conformational changes of DNA, we examined the intrinsic steady-state emission spectra of an 89-membered set of synthetic oligonucleotides with reported conformation (G-quadruplexes, i-motifs, single- and double stranded DNA) by means of multivariate analysis. Specifically, principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, albeit without discrimination between single- and double-stranded structures. Linear discriminant analysis of the same training set was exploited for the assessment of new sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labelling agent or dye, avoiding the related intrinsic bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5′-d[(G3T)3G3]-3′ (G3T), the most fluorescent G4 structure reported to date. This property is likely to arise from the similar base-stacking geometry in both types of structures.

scholarly journals FTIR Microspectroscopy Coupled with Two-Class Discrimination Segregates Markers Responsible for Inter- and Intra-Category Variance in Exfoliative Cervical Cytology

2008 ◽  
Vol 3 ◽  
pp. BMI.S592 ◽  
Author(s):  
Michael J. Walsh ◽  
Maneesh N. Singh ◽  
Helen F. Stringfellow ◽  
Hubert M. Pollock ◽  
Azzedine Hammiche ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Cell Types ◽  
Cervical Cytology ◽  
Low Grade ◽  
High Grade ◽  
Linear Discriminant ◽  
Ftir Microspectroscopy ◽  
Category Variance ◽  
Different Cell Types

Infrared (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a “biochemical fingerprint” of a particular cell type. Biomolecules absorb in the mid-IR (2–20 μm) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm–1 to 1,100 cm–1 spectral region; this included proteins (1,460 cm–1), glycoproteins (1,380 cm–1), amide III (1,260 cm–1), asymmetric (νas) PO2– (1,225 cm–1) and carbohydrates (1,155 cm–1). In contrast, symmetric (νs) PO2–(1,080 cm–1) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile.

scholarly journals RIGHT-ANGLE FLUORESCENCE SPECTROSCOPY FOR DIFFERENTIATION OF DISTILLED ALCOHOLIC BEVERAGES

2013 ◽  
Vol 12 (2) ◽  
pp. 83-92 ◽  
Author(s):  
Veronika Uríčková ◽  
Jana Sádecká ◽  
Pavel Májek
Keyword(s):  
Fluorescence Spectroscopy ◽  
Emission Spectra ◽  
Principal Component ◽  
Hierarchical Cluster ◽  
Alcoholic Beverages ◽  
Front Face ◽  
Excitation Wavelength ◽  
Spectroscopic Techniques ◽  
Excitation Spectra ◽  
Linear Discriminant

Abstract Total luminescence and synchronous scanning fluorescence spectroscopic techniques were investigated for differentiating brandies from mixed wine spirits. The studies were performed on 16 brandies from 3 different producers and 30 mixed wine spirits from 5 different producers. Differentiation between samples was accomplished by multivariate data analysis methods (principal component analysis, hierarchical cluster analysis, and linear discriminant analysis). Correct classification was obtained using emission spectra (400-650 nm) recorded at excitation wavelength 390 nm, excitation spectra (225-460 nm) obtained at emission wavelength 470 nm and synchronous fluorescence spectra (200-700 nm) collected at wavelength interval 80 nm. These results indicate that right-angle fluorescence spectroscopy offers a promising approach for the authentication of brandies as neither sample preparation nor special qualification of the personnel are required, and data acquisition and analysis are relatively simple when compared to front-face technique.

Intrinsic fluorescence–based label-free detection of bovine amyloid A amyloidosis

2021 ◽  
pp. 104063872110492
Author(s):  
Naoki Ujike ◽  
Susumu Iwaide ◽  
Yuki Ono ◽  
Takayuki Okano ◽  
Tomoaki Murakami
Keyword(s):  
Analytical Data ◽  
Principal Component ◽  
Histochemical Staining ◽  
Intrinsic Fluorescence ◽  
Label Free ◽  
Simple Method ◽  
Spectrometry Analysis ◽  
Amyloid A ◽  
Label Free Detection ◽  
Tissue Homogenates

Amyloidosis is diagnosed by the histologic detection of amyloid deposits; however, this method has limitations such as a prolonged diagnosis time and the need for histologic proficiency. We aimed to develop a rapid and simple method for diagnosing amyloidosis by targeting amyloid-specific endogenous fluorescence, which has not been reported previously, to our knowledge. Fluorescence fingerprint analysis of amyloid extracts and tissue homogenates derived from amyloid A (AA) amyloidosis-affected cattle exhibited a specific intrinsic fluorescence pattern. Furthermore, principal component analysis using analytical data revealed that AA could be identified by peaks near λex 350 nm and λem 430 nm. Fluorescence spectrometry analysis using tissue homogenates, which does not require special histochemical staining, enables the rapid detection of bovine AA.

The feasibility of multimodal fiber optic spectroscopy analysis in bladder cancer detection, grading, and staging

2021 ◽  
pp. 039156032110070
Author(s):  
Simone Morselli ◽  
Enrico Baria ◽  
Riccardo Cicchi ◽  
Andrea Liaci ◽  
Arcangelo Sebastianelli ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Fiber Optic ◽  
Ex Vivo ◽  
Principal Component ◽  
Classification Algorithm ◽  
Low Grade ◽  
Label Free ◽  
Linear Discriminant ◽  
Cancer Management

Objective: To prove the feasibility of Multimodal Fiber Optic Spectroscopy (MFOS) analysis in bladder cancer (BCa) detection, grading, and staging. Materials and methods: Bladder specimens from patients underwent TURBT or TURP were recorded and analyzed with MFOS within 30 min from excision. In detail, our MFOS combined fluorescence, Raman spectroscopy, and diffuse reflectance. We used these optical techniques to collect spectra from bladder biopsies, then we compared the obtained results to gold standard pathological analysis. Finally, we developed a classification algorithm based on principal component analysis-linear discriminant analysis. Results: A total of 169 specimens were collected and analyzed from 114 patients, 40 (23.7%) healthy (from TURP), and 129 (76.3%) with BCa. BCa specimens were divided according to their grade—34 (26.4%) low grade (LG) and 95 (73.6%) high grade (HG) BCa—and stage—64 (49.6%) Ta, 45 (34.9%) T1, and 20 (15.5%) T2. MFOS-based classification algorithm correctly discriminated healthy versus BCa with 90% accuracy, HG versus LG with 83% accuracy. Furthermore, it assessed tumor stage with 75% accuracy for Ta versus T1, 85% for T1 versus T2, and 86% for Ta versus T2. Conclusions: Our preliminary results suggest that MFOS could be a reliable, fast, and label-free tool for BCa assessment, providing also grading and staging information. This technique could be applied in future for in vivo inspection as well as of ex vivo tissue biopsies. Thus, MFOS might improve urothelial cancer management. Further studies are required.

scholarly journals Diagnosing Hirschsprung disease by detecting intestinal ganglion cells using label-free hyperspectral microscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marcos A. Soares de Oliveira ◽  
Laura Galganski ◽  
Sarah Stokes ◽  
Che -Wei Chang ◽  
Christopher D. Pivetti ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Second Harmonic ◽  
Hirschsprung Disease ◽  
Principal Component ◽  
Distal Colon ◽  
Support Vector ◽  
Label Free ◽  
Accurate Identification ◽  
Linear Discriminant ◽  
Multiple Biopsies

AbstractHirschsprung disease (HD) is a congenital disorder in the distal colon that is characterized by the absence of nerve ganglion cells in the diseased tissue. The primary treatment for HD is surgical intervention with resection of the aganglionic bowel. The accurate identification of the aganglionic segment depends on the histologic evaluation of multiple biopsies to determine the absence of ganglion cells in the tissue, which can be a time-consuming procedure. We investigate the feasibility of using a combination of label-free optical modalities, second harmonic generation (SHG); two-photon excitation autofluorescence (2PAF); and Raman spectroscopy (RS), to accurately locate and identify ganglion cells in murine intestinal tissue without the use of exogenous labels or dyes. We show that the image contrast provided by SHG and 2PAF signals allows for the visualization of the overall tissue morphology and localization of regions that may contain ganglion cells, while RS provides detailed multiplexed molecular information that can be used to accurately identify specific ganglion cells. Support vector machine, principal component analysis and linear discriminant analysis classification models were applied to the hyperspectral Raman data and showed that ganglion cells can be identified with a classification accuracy higher than 95%. Our findings suggest that a near real-time intraoperative histology method can be developed using these three optical modalities together that can aid pathologists and surgeons in rapid, accurate identification of ganglion cells to guide surgical decisions with minimal human intervention.

scholarly journals The Conformational Structural Change of Soy Glycinin via Lactic Acid Bacteria Fermentation Reduced Immunoglobulin E Reactivity

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2969
Author(s):  
Zhen Liu ◽  
Yaqiong Wang ◽  
Yifei Liu ◽  
Qiuqin Zhang ◽  
Wei Li ◽  
...  
Keyword(s):  
Particle Size ◽  
Conformational Changes ◽  
Fluorescence Intensity ◽  
Immunoglobulin E ◽  
Principal Component ◽  
Surface Hydrophobicity ◽  
Intrinsic Fluorescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ige Reactivity ◽  
Α Helix

This study investigated the fermentation of isolated soy glycinin by using the Lactiplantibacillus plantarum B1-6 strain, its reduction effect on immunoglobulin E (IgE) reactivity, the relationship with protein aggregation/gelation state and conformational changes. Fermentation was performed under different glycinin concentrations (0.1%, 0.5%, 1% and 2%, w/v) and varied fermentation terminal pH levels (FT-pH) (pH 6.0, 4.5, 4.0 and 3.5). L. plantarum B1-6 showed potency in reducing immunoreactivity to 0.10–69.85%, as determined by a sandwich enzyme-linked immunosorbent assay. At a FT-pH of 6.0 and 4.5, extremely low IgE reactivity (0.1–22.32%) was observed. Fermentation resulted in a great increase (2.31–6.8-fold) in particle size and a loss of intensity in A3 and basic subunits. The conformation of glycinin was altered, as demonstrated by improved surface hydrophobicity (1.33–7.39-fold), decreased intrinsic fluorescence intensity and the α-helix structure. Among the four selected concentrations, glycinin at 1% (w/v, G-1) evolved the greatest particles during fermentation and demonstrated the lowest immunoreactivity. Principal component analysis confirmed that particle size, intrinsic fluorescence intensity, α-helix and ionic bond were closely related to immunoreactivity reduction.

Evaluation of Chemotherapeutic Activity of the Selected Bases’ Analogues of Nucleic Acids Supported by ab initio Various Quantum Chemical Calculations

2020 ◽  
Vol 16 (2) ◽  
pp. 93-103 ◽  
Author(s):  
Piotr Kawczak ◽  
Leszek Bober ◽  
Tomasz Bączek
Keyword(s):  
Biological Activity ◽  
Nucleic Acids ◽  
Ab Initio ◽  
Quantum Chemical Calculations ◽  
Quantum Chemical ◽  
Structural Parameters ◽  
Principal Component ◽  
Polarizable Continuum Model ◽  
Activity Data ◽  
Qsar Models

Background: Pharmacological and physicochemical classification of bases’ selected analogues of nucleic acids is proposed in the study. Objective: Structural parameters received by the PCM (Polarizable Continuum Model) with several types of calculation methods for the structures in vacuo and in the aquatic environment together with the huge set of extra molecular descriptors obtained by the professional software and literature values of biological activity were used to search the relationships. Methods: Principal Component Analysis (PCA) together with Factor Analysis (FA) and Multiple Linear Regressions (MLR) as the types of the chemometric approach based on semi-empirical ab initio molecular modeling studies were performed. Results: The equations with statistically significant descriptors were proposed to demonstrate both the common and differentiating characteristics of the bases' analogues of nucleic acids based on the quantum chemical calculations and biological activity data. Conclusion: The obtained QSAR models can be used for predicting and explaining the activity of studied molecules.

scholarly journals Real Time Breath Analysis Using Portable Gas Chromatography for Adult Asthma Phenotypes

Metabolites ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 265
Author(s):  
Ruchi Sharma ◽  
Wenzhe Zang ◽  
Menglian Zhou ◽  
Nicole Schafer ◽  
Lesa A. Begley ◽  
...  
Keyword(s):  
Gas Chromatography ◽  
Real Time ◽  
Rapid Assessment ◽  
Principal Component ◽  
Corticosteroid Treatment ◽  
Breath Analysis ◽  
Blood Eosinophil ◽  
Exhaled Breath ◽  
Respiratory Illnesses ◽  
Linear Discriminant

Asthma is heterogeneous but accessible biomarkers to distinguish relevant phenotypes remain lacking, particularly in non-Type 2 (T2)-high asthma. Moreover, common clinical characteristics in both T2-high and T2-low asthma (e.g., atopy, obesity, inhaled steroid use) may confound interpretation of putative biomarkers and of underlying biology. This study aimed to identify volatile organic compounds (VOCs) in exhaled breath that distinguish not only asthmatic and non-asthmatic subjects, but also atopic non-asthmatic controls and also by variables that reflect clinical differences among asthmatic adults. A total of 73 participants (30 asthma, eight atopic non-asthma, and 35 non-asthma/non-atopic subjects) were recruited for this pilot study. A total of 79 breath samples were analyzed in real-time using an automated portable gas chromatography (GC) device developed in-house. GC-mass spectrometry was also used to identify the VOCs in breath. Machine learning, linear discriminant analysis, and principal component analysis were used to identify the biomarkers. Our results show that the portable GC was able to complete breath analysis in 30 min. A set of nine biomarkers distinguished asthma and non-asthma/non-atopic subjects, while sets of two and of four biomarkers, respectively, further distinguished asthmatic from atopic controls, and between atopic and non-atopic controls. Additional unique biomarkers were identified that discriminate subjects by blood eosinophil levels, obese status, inhaled corticosteroid treatment, and also acute upper respiratory illnesses within asthmatic groups. Our work demonstrates that breath VOC profiling can be a clinically accessible tool for asthma diagnosis and phenotyping. A portable GC system is a viable option for rapid assessment in asthma.

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