Flavin Adenine Dinucleotide Research Articles

UVA Hyperspectral Light-Sheet Microscopy for Volumetric Metabolic Imaging: Application to Preimplantation Embryo Development.

Cellular metabolism is a key regulator of energetics, cell growth, regeneration, and homeostasis. Spatially mapping the heterogeneity of cellular metabolic activity is of great importance for unraveling the overall cell and tissue health. In this regard, imaging the endogenous metabolic cofactors, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), with subcellular resolution and in a noninvasive manner would be useful to determine tissue and cell viability in a clinical environment, but practical use is limited by current imaging techniques. In this paper, we demonstrate the use of phasor-based hyperspectral light-sheet (HS-LS) microscopy using a single UVA excitation wavelength as a route to mapping metabolism in three dimensions. We show that excitation solely at a UVA wavelength of 375 nm can simultaneously excite NAD(P)H and FAD autofluorescence, while their relative contributions can be readily quantified using a hardware-based spectral phasor analysis. We demonstrate the potential of our HS-LS system by capturing dynamic changes in metabolic activity during preimplantation embryo development. To validate our approach, we delineate metabolic changes during preimplantation embryo development from volumetric maps of metabolic activity. Importantly, our approach overcomes the need for multiple excitation wavelengths, two-photon imaging, or significant postprocessing of data, paving the way toward clinical translation, such as in situ, noninvasive assessment of embryo viability.

High levels of FAD autofluorescence indicate pathology preceding cell death

Flavin adenine dinucleotide (FAD) autofluorescence from cells reports on the enzymatic activity which involves FAD as a cofactor. Most of the cellular FAD fluorescence comes from complex II of the electron transport chain in mitochondria and can be assessed with inhibitor analysis. The intensity of FAD autofluorescence is not homogeneous and vary between cells in tissue and in cell culture types. Using primary co-culture of neurons and astrocytes, and human skin fibroblasts we have found that very high FAD autofluorescence is a result of an overactivation of the mitochondrial complex II from ETC and from the activity of monoamine oxidases. Cells with high FAD autofluorescence were mostly intact and were not co-labelled with indicators for necrosis or apoptosis. However, cells with high FAD fluorescence showed activation of apoptosis and necrosis within 24 h after initial measurements. Thus, high level of FAD autofluorescence is an indicator of cell pathology and reveals an upcoming apoptosis and necrosis.

Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Impacts of high-dose riboflavin on cytotoxicity, antioxidant, growth, reproductive gene expressions, and genotoxicity in the rainbow trout gonadal cells

Riboflavin (vitamin B2 found in food) is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which study as coenzymes for a variety of cellular processes including biosynthesis, homocysteine metabolism, detoxification, and various oxidation and reduction reactions. Although studies on the symptoms resulting from riboflavin deficiency are intense, studies on the effects of high doses of riboflavin are almost absent. This report aimed to examine the actions of riboflavin on cell viability, the transcriptional expressions of antioxidant enzyme (gsr and gpx1a), growth (gh1, igf1, and igf2), the reproductive (bol) genes and DNA damage in the rainbow trout gonad cells (RTG-2) for 48 h. All concentrations of riboflavin (3.125, 6.25, 12.5, 25, 50, and 100 μM) significantly reduced the RTG-2 cell viability. Riboflavin (LD50: 12.5 μM) significantly downregulated the transcriptional expressions of gpx1a, igf1, and bol genes, while it non-significantly upregulated or downregulated the transcriptional expression of gsr, igf2, and gh1 genes in the RTG-2 cells in comparison to the control group for 48 h. The comet assay demonstrated that riboflavin significantly raised tail DNA% >10% DMSO (positive control). Based on the outcomes, high doses of riboflavin exhibit the potential to have a role in cellular mechanisms, including especially reproduction, DNA damage, and cell death.

Human Saliva as a Substitute Diagnostic Medium for the Detection of Oral Lesions Using the Stokes Shift Spectroscopy: Discrimination among the Groups by Multivariate Analysis Methods.

Our objective in the present study is to detect oral mucosal lesions non-invasively by probing two solutions with reference to diagnostic technique and non-invasive media. In the diagnostic technique, Stokes shift (SS) spectroscopy (SSS) has been utilized for the detection of oral lesions. In the diagnostic media, human oral tissue and saliva are included. SS measurements are carried out on oral squamous cell carcinoma (OSCC), dysplastic (precancer), and normal/control tissue and saliva samples. Measurements are performed on 86 tissue and 86 saliva samples using the commercially available spectrofluorometer. Offset wavelength of 120 nm, which is the Stoke shift of nicotinamide adenine dinucleotide (NADH) has been selected over the other offsets (i.e., 20, 40, 70 and 90 nm). Presence of tryptophan, collagen, NADH, and flavin adenine dinucleotide (FAD) bands were noticed in the SS spectra of tissue. Like the tissue spectra, presence of these bands was also found in the SS spectra of saliva except the collagen band. Classification among the samples accomplished by the make use of multivariate analysis methods. In the multivariate analysis methods, principal component analysis (PCA) is applied first on SS data of tissue and saliva and then Mahalanobis distance (MD) model and receiver operating characteristic (ROC) analysis employed successively. Overall accuracy values of 94.91 %, 84.61 %, and 85.24 % were obtained among OSCC to normal, dysplasia to normal, and OSCC to dysplasia for tissue samples and 88.46 %, 90.16 % and 94.91 % accuracy values were obtained for saliva using the SS spectroscopy. Obtained results of human saliva are equivalent to human oral tissue using the SS spectroscopy. It indicates that saliva may be utilized as a substitute diagnostic medium and SS spectroscopy as a diagnostic technique for non-invasive detection of oral lesions at the primarily stage.

A marine cryptochrome with an inverse photo-oligomerization mechanism

Cryptochromes (CRYs) are a structurally conserved but functionally diverse family of proteins that can confer unique sensory properties to organisms. In the marine bristle worm Platynereis dumerilii, its light receptive cryptochrome L-CRY (PdLCry) allows the animal to discriminate between sunlight and moonlight, an important requirement for synchronizing its lunar cycle-dependent mass spawning. Using cryo-electron microscopy, we show that in the dark, PdLCry adopts a dimer arrangement observed neither in plant nor insect CRYs. Intense illumination disassembles the dimer into monomers. Structural and functional data suggest a mechanistic coupling between the light-sensing flavin adenine dinucleotide chromophore, the dimer interface, and the C-terminal tail helix, with a likely involvement of the phosphate binding loop. Taken together, our work establishes PdLCry as a CRY protein with inverse photo-oligomerization with respect to plant CRYs, and provides molecular insights into how this protein might help discriminating the different light intensities associated with sunlight and moonlight.

Long-Time Oxygen and Superoxide Localization in Arabidopsis thaliana Cryptochrome.

Cryptochromes are proteins that are highly conserved across species and in many instances bind the flavin adenine dinucleotide (FAD) cofactor within their photolyase-homology region (PHR) domain. The FAD cofactor has multiple redox states that help catalyze reactions, and absorbs photons at about 450 nm, a feature linked to the light-related functions of cryptochrome proteins. Reactive oxygen species (ROS) are produced from redox reactions involving molecular oxygen and are involved in a myriad of biological processes. Superoxide O2•- is an exemplary ROS that may be formed through electron transfer from FAD to O2, generating an electron radical pair. Although the formation of a superoxide-FAD radical pair has been speculated, it is still unclear if the required process steps could be realized in cryptochrome. Here, we present results from molecular dynamics (MD) simulations of oxygen interacting with the PHR domain of Arabidopsis thaliana cryptochrome 1 (AtCRY1). Using MD simulation trajectories, oxygen binding locations are characterized through both the O2-FAD intermolecular distance and the local protein environment. Oxygen unbinding times are characterized through replica simulations of the bound oxygen. Simulations reveal that oxygen molecules can localize at certain sites within the cryptochrome protein for tens of nanoseconds, and superoxide molecules can localize for significantly longer. This relatively long-duration molecule binding suggests the possibility of an electron-transfer reaction leading to superoxide formation. Estimates of electron-transfer rates using the Marcus theory are performed for the identified potential binding sites. Molecular oxygen binding results are compared with recent results demonstrating long-time oxygen binding within the electron-transfer flavoprotein (ETF), another FAD binding protein.

Photo-enzyme-polymerized hydrogel platform exhibits photo-switchable redox reversibility for diabetic wound healing

Redox homeostasis catalyzed by series oxidoreductases is important to maintain normal physiological functions, including mutually contradictory reactive oxygen species generation or reduction by separated enzyme regulation during cellular metabolism. The unidirectional enzyme regulation of redox homeostasis by biocatalytic reaction is an emerging catalytic medicine approach to disease biomimetic treatments. Bioinspired by photosynthesis, an efficient photo-enzyme-coupled hydrogel platform (Gel) with a spatio-temporally controlled hydrogel network was constructed through photoenzymatic-initiated radical polymerization in the absence of the corresponding enzyme substrate. Then, the coupling mechanism of light-induced electron transfer-enzyme activity center allosteric was explored through free radical analysis and theoretical calculations. The photo-switchable redox performance has been demonstrated based on the special anaerobic dehydrogenases, the flavin adenine dinucleotide (FAD)-centered dihydrolipoamide dehydrogenase (DLD) platform. At last, both in vitro and in vivo biological effects have been verified to evaluate the bidirectional oxidation/antioxidant modulation for the complicated wound healing in diabetic mice and wound-infected animals. The photoenzymatic coupling dehydrogenase-laden hydrogel platform proposed in this work provides ideas for the engineering design of enzymes, and the physical-biochemical regulatory mechanisms also offers a theoretical basis for controllable activation of enzyme activity, allowing for potential biomedical applications in metabolic processes.

Riboflavin interactions with the chicken isolated carrier protein

Riboflavin, a member of the B vitamin family, is a water-soluble vitamin that participates in energy metabolism processes via two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), in oxidized and reduced forms. Low levels of riboflavin have been associated with growth and developmental problems. In an effort to investigate the role of hydrogen bonding in the interactions between riboflavin and chicken riboflavin binding protein, the solid state geometry characteristics of a riboflavin derivative stripped of hydroxyl groups except the primary one, N-(6′-hydroxyhexyl)isoalloxazine, were investigated and found that π-stacking and hydrogen bonding involving the isoalloxazine rings are the primary intermolecular interactions. Subsequent comparative fluorescence studies showed that at neutral pH, in presence of the protein, quenching of N-(6′-hydroxyhexyl)isoalloxazine and riboflavin occurred similarly suggesting that the hydroxyl groups were not a key component of the vitamin protein interactions in the binding pocket.

A raman spectroscopic investigation of conformation of flavin adenine dinucleotide, a coenzyme of d-amino acid oxidase

Based on silver nanoparticles application, surface-enhanced Raman scattering spectra of D-amino acid oxidase from pig kidney were recorded and analyzed. Spectral parameters characteristic of conformational changes in cofactor flavin adenine dinucleotide during activation of the enzyme by D-amino acids were revealed. It was found that enzyme substrate specificity determines the amount of time from the start of of the conformational changes of flavin adenine dinucleotide until they no longer occur: in the presence of D-alanine, registration of the said conformational changes takes up just a few seconds, while it takes 10 min in the presence of D-serine .

The IbeA protein from adherent invasive Escherichia coli isa flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

Discovery of a Novel Cellobiose Dehydrogenase from Cellulomonas palmilytica EW123 and Its Sugar Acids Production.

Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 (CpCDH) was cloned and characterized. The CpCDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in CpCDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of CpCDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of CpCDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The CpCDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, CpCDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of CpCDH for cellobiose and lactose were 2.05 x 105 and 9.06 x 104 (M-1 s-1), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of CpCDH. This study establishes CpCDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome

An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH−) in CraCRY and related (6–4) photolyases. The thermodynamic stability of FADH− remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH− varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH− suggests an essential role of 8-HDF in the bifunctionality of CraCRY.

A Raman Spectroscopic Study of the Conformation of Flavin Adenine Dinucleotide, a CoEnzyme of D-Amino Acid Oxidase

A Raman Spectroscopic Study of the Conformation of Flavin Adenine Dinucleotide, a CoEnzyme of D-Amino Acid Oxidase

Effect of Fermentation Scale on Microbiota Dynamics and Metabolic Functions for Indigo Reduction.

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively.

Widespread Distribution of the arsO Gene Confers Bacterial Resistance to Environmental Antimony.

Microbial oxidation of environmental antimonite (Sb(III)) to antimonate (Sb(V)) is an antimony (Sb) detoxification mechanism. Ensifer adhaerens ST2, a bacterial isolate from a Sb-contaminated paddy soil, oxidizes Sb(III) to Sb(V) under oxic conditions by an unknown mechanism. Genomic analysis of ST2 reveals a gene of unknown function in an arsenic resistance (ars) operon that we term arsO. The transcription level of arsO was significantly upregulated by the addition of Sb(III). ArsO is predicted to be a flavoprotein monooxygenase but shows low sequence similarity to other flavoprotein monooxygenases. Expression of arsO in the arsenic-hypersensitive Escherichia coli strain AW3110Δars conferred increased resistance to Sb(III) but not arsenite (As(III)) or methylarsenite (MAs(III)). Purified ArsO catalyzes Sb(III) oxidation to Sb(V) with NADPH or NADH as the electron donor but does not oxidize As(III) or MAs(III). The purified enzyme contains flavin adenine dinucleotide (FAD) at a ratio of 0.62 mol of FAD/mol protein, and enzymatic activity was increased by addition of FAD. Bioinformatic analyses show that arsO genes are widely distributed in metagenomes from different environments and are particularly abundant in environments affected by human activities. This study demonstrates that ArsO is an environmental Sb(III) oxidase that plays a significant role in the detoxification of Sb(III).

Erweiterung des Reaktionsspektrums: Nachweis des 5‐Deazaflavin‐Radikals durch photochemisch induzierte dynamische Kernpolarisation

AbstractDeazaflavine sind wichtige Analoga der natürlich vorkommenden Flavine, Riboflavin, Flavinmononukleotid (FMN) und Flavin‐Adenin‐Dinukleotid (FAD). Durch Austausch des natürlichen Kofaktors mit dem entsprechenden 5‐Deazaflavin konnten in einer Reihe von Flavoproteinen wichtige Erkenntnisse zum Reaktionsmechanismus gewonnen sowie deren Reaktivität beeinflusst werden. Es wurde häufig beobachtet, dass Ein‐Elektronentransfer‐Reaktionen in Flavoproteinen mit 5‐Deazaflavin als Kofaktor eingeschränkt sind. Hieraus wurde gefolgert, dass der im Vergleich mit dem entsprechenden Flavinsemichinon geringeren Stabilität des 5‐Deazaflavin‐Radikals rasch ablaufende Reoxidations‐ und Disproportionierungsreaktionen zugrunde liegen. Das vermeintliche Paradigma, wonach 5‐Deazaflavin lediglich als Zwei‐Elektronen‐ bzw. Hydrid‐Akzeptor/Donor fungiert, sozusagen ein “nicotinamide in flavin clothing” darstellt, muss nun durch die unter biologisch relevanten Reaktionsbedingungen erfolgte indirekte Beobachtung einer einfach reduzierten (paramagnetischen) Spezies mit Hilfe der 1H‐photo‐CIDNP‐NMR (photo‐CIDNP: photo‐chemisch induzierte dynamische Kernpolarisation) neu bewertet werden.

Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization.

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor-"a nicotinamide in flavin clothing"-needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) 1 H nuclear magnetic resonance (NMR) under biologically relevant conditions.

Possible role of essential tyrosine on the protonation of isoalloxazine ring in the excited singlet state and the triplet state in photolyase from Escherichia coli. Molecular orbital study

Photolyases from Escherichia coli. (PLEC) function to repair UV-damaged DNA. Photolyases and cryptochromes (altogether 42 proteins) are found to contain at least one tyrosine (Tyr) residue near the isoalloxazine ring (Iso) of flavin adenine dinucleotide (FAD) within 1.2 nm, Tyr281 in PLEC. Iso in PLEC is surrounded by four Trp residues and one Tyr within 1.2 nm from Iso. Photoinduced charge transfer interactions (PCT) among these chromophores in the Iso-Tyr281-Trp277-Trp338-Trp382-Trp384 system were studied by means of a semi-empirical molecular orbital (MO) method. Structure of the system was extracted from crystal structure of PLEC. In the ground state the PCT interactions between any pairs were negligible. Upon photo-excitation almost one electron transferred from Trp382 to the excited Iso, to which an electron transport chain from Tyr residues at the protein - water interface to Iso is connected. Protonation at N1 or N5 of Iso from Tyr281 markedly stabilized the transition energy in the excited singlet state and triplet state, depending on the polarity of the medium, but not from a source other than Tyr281. In these states, nearly one electron migrates from Tyr281 to Trp382. Effects of the protonation from Tyr281 to the fully reduced Iso were also examined, where total charge of the system was −2. In the ground state the energy of the system was stabilized by the protonation, when the proton was bound to N5 of Iso, but not to N1. In the excited singlet state and triplet state of the fully reduced Iso, the transition energies of the system were also stabilized upon the protonation to N1 or N5 of Iso from Tyr281, but not from a source other than Tyr281. In the ground state almost two negative charges were located at Iso. In the excited singlet state one of two negative charges was mostly localized to Iso and another to Trp277 at the dielectric constant, 3, and to Trp384 at the dielectric constant, 10. In the triplet state without the protonation, one of two negative charges was localized to Iso and another to Trp384 at any dielectric constant. Upon protonation to N1 or N5 of Iso in the triplet state, one of the negative charges was localized to Iso and another to Tyr281 at any value of dielectric constant. Thus Tyr281 closely interacted with Iso in the excited singlet state and triplet state, and other Trp residues in the system through the proton transfer.

Laser Induced Fluorescence Spectroscopy analysis of kidney tissues: A pilot study for the identification of renal cell carcinoma.

The 325nm-excited autofluorescence spectra from cancerous and normal renal tissues were collected ex-vivo biopsy tissue samples, through an optical fibre probe-based system. Noticeable changes in intensity/wavelength were observed in the fluorescence emissions from endogenous fluorophores such as collagen, Nicotinamide adenine dinucleotide(NADH), Vitamin A(retinol) and Flavin adenine dinucleotide(FAD), in pathological conditions with respect to the normal state. The energy metabolism involved in clear cell renal cell carcinoma(ccRCC) and chromophobe renal cell carcinoma (chRCC) are reflected in the fluorescence emission band at 445 nm due to bound NADH attributed to enhanced oxidative phosphorylation in chRCC and emission at 465 nm contributed by free NADH showing higher glycolytic action in ccRCC. The principal component analysis and one-way ANOVA effectively discriminate ccRCC from chRCC. It is shown that laser induced fluorescence technique with 325 nm excitation can be a suitable technique for optical pathology and in vivo surgical boundary demarcation in renal cell carcinoma. This article is protected by copyright. All rights reserved.