Analyses of pre-steady-state kinetics and isotope effects of the γ-elimination reaction catalyzed by Citrobacter freundii methionine γ-lyase.

Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5'-phosphate-dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and ß-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.

Modulation of Molecular Structure and Mechanical Properties of κ-Carrageenan-Gelatin Hydrogel with Multi-Walled Carbon Nanotubes.

Hydrogels, three-dimensional hydrophilic water-insoluble polymer networks having mechanical properties inherent for solids, have attracted continuous research attention over a long time period. Here, we studied the structure and properties of hydrogel based on gelatin, κ-carrageenan and CNTs using the combination of SAXS, PXRD, AFM microscopy, SEM and rheology methods. We have shown that the integration of polysaccharide and protein in the composite hydrogel leads to suppression of their individual structural features and homogenization of two macromolecular components into a single structural formation. According to obtained SAXS results, we observed the supramolecular complex, which includes both polysaccharide and protein components associated with each other. It was determined that hydrogel structure formed in the initial solution state (dispersion) retains hydrogel supramolecular structure under its cooling up to gel state. The sizes of dense cores of these polyelectrolyte complexes (PEC) slightly decrease in the gel state in comparison with PEC water dispersion. The introduction of CNTs to hydrogel does not principally change the type of supramolecular structure and common structural tendencies observed for dispersion and gel states of the system. It was shown that carbon nanotubes embedded in hydrogel act as the supplementary template for formation of the three-dimensional net, giving additional mechanical strengthening to the studied system.

Serine 51 residue of Citrobacter freundii tyrosine phenol-lyase assists in C-α-proton abstraction and transfer in the reaction with substrate.

In the spatial structure of tyrosine phenol-lyase, the Ser51 residue is located in the active site of the enzyme. The replacement of Ser51 with Ala by site-directed mutagenesis led to a decrease of the kcat/Km parameter for reactions with l-tyrosine and 3-fluoro-l-tyrosine by three orders of magnitude, compared to wild type enzyme. For the elimination reactions of S-alkylcysteines, the values of kcat/Km decreased by an average of two orders of magnitude. The results of spectral studies of the mutant enzyme gave evidence for a considerable change of the chiral properties of the active site as a result of the replacement. Fast kinetic studies for the complexes of the mutant form with competitive inhibitors allowed us to conclude that the Ser51 residue interacts with the side chain amino group of Lys257 at the stage of C-α-proton abstraction. This interaction ensures the correct orientation of the side chain of Lys257 accepting the C-α-proton of the external aldimine and stabilizes its ammonium form. Also, it is probable that Ser51 takes part in formation of a chain of hydrogen bonds which is necessary to perform the transfer of the C-α-proton to the C-4'-position of the leaving phenol group in the reaction with the natural substrate.

The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.

In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and ß-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-ß- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and ß-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors.

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the ß-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

A straightforward kinetic evidence for coexistence of "induced fit" and "selected fit" in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris.

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme ("induced fit") is realized in the case of mutant Y72F enzyme reaction with OIA. For the reaction of mutant enzyme with l-Trp at lower concentrations of the latter a principally different kinetic scheme is observed. This scheme suggests that binding of the substrate and formation of the quinonoid intermediate are at fast equilibrium, while preceding conformational changes of the holoenzyme proceed with measureable rates ("selected fit"). For the reaction with S-ethyl-l-cysteine the observed concentration dependence of kobs agrees with the realization of both kinetic schemes, the "selected fit" becoming predominant at lower concentrations of substrate, the "induced fit"- at higher ones. In the reaction with S-ethyl-l-cysteine the formation of the quinonoid intermediate proceeds slower than does catalytic α,ß-elimination of ethylthiol from S-ethyl-l-cysteine, and consequently does not play a considerable role in the catalysis, which may be effected by a concerted E2 mechanism.

The role of substrate strain in the mechanism of the carbon-carbon lyases.

The carbon-carbon lyases, tryptophan indole lyase (TIL) and tyrosine phenol-lyase (TPL) are bacterial enzymes which catalyze the reversible elimination of indole and phenol from l-tryptophan and l-tyrosine, respectively. These PLP-dependent enzymes show high sequence homology (∼40% identity) and both form homotetrameric structures. Steady state kinetic studies with both enzymes show that an active site base is essential for activity, and α-deuterated substrates exhibit modest primary isotope effects on kcat and kcat/Km, suggesting that substrate deprotonation is partially rate-limiting. Pre-steady state kinetics with TPL and TIL show rapid formation of external aldimine intermediates, followed by deprotonation to give quinonoid intermediates absorbing at about 500nm. In the presence of phenol and indole analogues, 4-hydroxypyridine and benzimidazole, the quinonoid intermediates of TPL and TIL decay to aminoacrylate intermediates, with λmax at about 340nm. Surprisingly, there are significant kinetic isotope effects on both formation and subsequent decay of the quinonoid intermediates when α-deuterated substrates are used. The crystal structure of TPL with a bound competitive inhibitor, 4-hydroxyphenylpropionate, identified several essential catalytic residues: Tyr-71, Thr-124, Arg-381, and Phe-448. The active sites of TIL and TPL are highly conserved with the exceptions of these residues: Arg-381(TPL)/Ile-396 (TIL); Thr-124 (TPL)/Asp-137 (TIL), and Phe-448 (TPL)/His-463 (TIL). Mutagenesis of these residues results in dramatic decreases in catalytic activity without changing substrate specificity. The conserved tyrosine, Tyr-71 (TPL)/Tyr-74 (TIL) is essential for elimination activity with both enzymes, and likely plays a role as a proton donor to the leaving group. Mutation of Arg-381 and Thr-124 of TPL to alanine results in very low but measurable catalytic activity. Crystallography of Y71F and F448H TPL with 3-fluoro-l-tyrosine bound demonstrated that there are two quinonoid structures, relaxed and tense. In the relaxed structure, the substrate aromatic ring is in plane with the Cß-Cγ bond, but in the tense structure, the substrate aromatic ring is about 20° out of plane with the Cß-Cγ bond. In the tense structure, hydrogen bonds are formed between the substrate OH and the guanidinium of Arg-381 and the OH of Thr-124, and the phenyl rings of Phe-448 and 449 provide steric strain. Based on the effects of mutagenesis, the substrate strain is estimated to contribute about 10(8) to TPL catalysis. Thus, the mechanisms of TPL and TIL require both substrate strain and acid/base catalysis, and substrate strain is probably responsible for the very high substrate specificity of TPL and TIL.

Crystal structure of the external aldimine of Citrobacter freundii methionine γ-lyase with glycine provides insight in mechanisms of two stages of physiological reaction and isotope exchange of α- and ß-protons of competitive inhibitors.

The three-dimensional structure of the external aldimine of Citrobacter freundii methionine γ-lyase with competitive inhibitor glycine has been determined at 2.45 Å resolution. It revealed subtle conformational changes providing effective binding of the inhibitor and facilitating labilization of Cα-protons of the external aldimine. The structure shows that 1, 3-prototropic shift of Cα-proton to C4'-atom of the cofactor may proceed with participation of active site Lys210 residue whose location is favorable for performing this transformation by a concerted mechanism. The observed stereoselectivity of isotopic exchange of enantiotopic Cα-protons of glycine may be explained on the basis of external aldimine structure. The exchange of Cα-pro-(R)-proton of the external aldimine might proceed in the course of the concerted transfer of the proton from Cα-atom of glycine to C4'-atom of the cofactor. The exchange of Cα-pro-(S)-proton may be performed with participation of Tyr113 residue which should be present in its basic form. The isotopic exchange of ß-protons, which is observed for amino acids bearing longer side groups, may be effected by two catalytic groups: Lys210 in its basic form, and Tyr113 acting as a general acid.

Bilateral and multifocal Warthin's tumor of parotid gland: two case reports and review of literature.

Warthin's tumor is the second most common benign neoplasm of the parotid. Most of cases are represented by a single localization, while only a small percentage of patients presents bilateral lesions or unilateral multifocal pattern. Warthin's tumor has an excellent prognosis due to the low rate of recurrence after surgical treatment. Malignant transformation occurs in less than 1% of cases. The aim of this article is to present two unusual cases of Warthin's tumor and an updated review of the latest scientific literature.

A complication following tooth extraction: chronic suppurative osteomyelitis.

UNLABELLED: A complication following tooth extraction: a case report of chronic suppurative osteomyelitis. OBJECTIVE: This article presents a case report about the surgery treatment of chronic suppurative osteomyelitis following a tooth extraction. METHODS: Cone beam computed tomography revealed a sequestrum bone formation that required the sequestrectomy and the debridement of the involved area. The prescription of oral penicillin and metronidazole were necessary after and before the surgery. Also 20 sessions of hyperbaric oxygen therapy were important for the healing of the marrow space. RESULTS: The histologic test confirmed the diagnosis of "Chronic suppurative osteomyelitis". Clinically the post-operative course showed no complications but a good healing of the bone tissue. Culture reports revealed two microorganisms, streptococcus viridans and staphylococcus, that are sensitive to penicillin. CONCLUSIONS: Clinical results confirmed the validity of the sequestrectomy and the debridement of the involved area for the treatment of chronic suppurative osteomyelitis. Such approach has always to be preferred because it guarantees the healing of bone tissue.

Crystallographic snapshots of tyrosine phenol-lyase show that substrate strain plays a role in C-C bond cleavage.

The key step in the enzymatic reaction catalyzed by tyrosine phenol-lyase (TPL) is reversible cleavage of the Cß-Cγ bond of L-tyrosine. Here, we present X-ray structures for two enzymatic states that form just before and after the cleavage of the carbon-carbon bond. As for most other pyridoxal 5'-phosphate-dependent enzymes, the first state, a quinonoid intermediate, is central for the catalysis. We captured this relatively unstable intermediate in the crystalline state by introducing substitutions Y71F or F448H in Citrobacter freundii TPL and briefly soaking crystals of the mutant enzymes with a substrate 3-fluoro-L-tyrosine followed by flash-cooling. The X-ray structures, determined at ~2.0 Å resolution, reveal two quinonoid geometries: "relaxed" in the open and "tense" in the closed state of the active site. The "tense" state is characterized by changes in enzyme contacts made with the substrate's phenolic moiety, which result in significantly strained conformation at Cß and Cγ positions. We also captured, at 2.25 Å resolution, the X-ray structure for the state just after the substrate's Cß-Cγ bond cleavage by preparing the ternary complex between TPL, alanine quinonoid and pyridine N-oxide, which mimics the α-aminoacrylate intermediate with bound phenol. In this state, the enzyme-ligand contacts remain almost exactly the same as in the "tense" quinonoid, indicating that the strain induced by the closure of the active site facilitates elimination of phenol. Taken together, structural observations demonstrate that the enzyme serves not only to stabilize the transition state but also to destabilize the ground state.

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery.

Kinetics, biodistribution, and histological studies were performed to evaluate the particle-size effects on the distribution of 15 nm and 50 nm PEG-coated colloidal gold (CG) particles and 160 nm silica/gold nanoshells (NSs) in rats and rabbits. The above nanoparticles (NPs) were used as a model because of their importance for current biomedical applications such as photothermal therapy, optical coherence tomography, and resonance-scattering imaging. The dynamics of NPs circulation in vivo was evaluated after intravenous administration of 15 nm CG NPs to rabbit, and the maximal concentrations of gold were observed 15-30 min after injection. Rats were injected in the tail vein with PEG-coated NPs (about 0.3 mg Au/kg rats). 24 h after injection, the accumulation of gold in different organs and blood was determined by atomic absorption spectroscopy. In accordance with the published reports, we observed 15 nm particles in all organs with rather smooth distribution over liver, spleen and blood. By contrast, the larger NSs were accumulated mainly in the liver and spleen. For rabbits, the biodistribution was similar (72 h after intravenous injection). We report also preliminary data on the light microscopy and TEM histological examination that allows evaluation of the changes in biotissues after gold NPs treatment.

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates.

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.

State of the art on autologous mesothelial transplant in animals and humans.

Sixteen years ago rabbit and human mesothelial cells were successfully cultured and autoimplanted. The aim of the study was merely to demonstrate that mesothelial implant was possible and interesting not only in peritoneal dialysis, but also in the vaster field of medicine and surgery concerning all the mesothelial districts of the body. The aim of this paper is to recollect the steps which have led to autologous mesothelial transplantation and verify if the technique has been validated and adopted by others. Review of the literature published in the last 15 years shows that intraperitoneal transplantation of mesothelial cells has been effective in reducing the formation of peritoneal adhesions, and in remodeling the area of mesothelial denudation. New studies on the mesothelial cell opened the way to construction of transplantable tissue-engineered artificial peritoneum, to the utilization of mesothelial progenitor cells and to find simple methods to collect autologous mesothelial cells. Finally mesothelial transplantation may represent a new neovascular therapy in the prevention and treatment of ischemic coronary heart disease.

Possible role of milky spots in mesothelial transplantation.

Milky spots are very small omental organs, in contact with peritoneal membrane, devoid of capsule and consisting of macrophages, lymphocytes and a few plasma cells supported by blood and lymphatic vessels. The exact role of these particular organs is still not clear, but they are similar to lymphatic structures and it is clear that they play a role in peritoneal infection and abdominal tumors. Peritoneal dialysis seems to activate the milky spots changing their morphology. The authors try to formulate some hypotheses on the role played by these little omental organs during autologous mesothelial transplant.

Evaluation of exercise testing for subjects with isolated proteinuria and/or hematuria of unclear significance.

BACKGROUND: A frequent problem that family doctors face is the meaning of small quantities of blood or protein in urine samples. Patients with this problem are often either neglected or referred to specialists for complex, expensive, and often invasive diagnostic procedures. Exercise testing has never been considered in nephrology, except for some attempts in diabetic patients. METHODS: We report on a study conducted over 12 years with patients referred for slight hematuria and/or proteinuria to determine whether exercise testing could be a diagnostic aid in some or all of them. We performed exercise testing using a treadmill preceded and followed by urine analysis, with a kidney biopsy within 10 days. Of the 94 patients enrolled in the study, only those with a positive exercise test turned out to have parenchymal nephropathy. At the end of the study, we simplified the quantification of exertion, dispensing with the treadmill and drastically reducing the number of urinary parameters considered. RESULTS: In patients with histological evidence of kidney damage, most of the variables increased significantly after the test. Statistical analysis also showed that determination of proteinuria and hematuria alone guaranteed maximum predictability. We found that it is also possible to simplify the quantification of effort/exertion and to drastically reduce the number of urinary parameters and still obtain significant results. CONCLUSIONS: Exercise testing provides useful information about the significance of microhematuria and proteinuria, reducing the number of cases that need to be referred to specialists. The method needs to be validated in other studies, but our results suggest that family doctors could use simple dipsticks to screen the many cases of microhematuria or proteinuria observed in daily practice. The method seems useful in eliminating doubts and unnecessary diagnostic costs.

High doses of water increase the purifying capacity of the kidneys.

BACKGROUND: In previous studies we were successful in demonstrating that the administration of water over a short period of time increases the transport capacity in the excretory tract of rabbit ureters by increasing urinary volume in the ureter from 0.3 ml/min to 10 ml/min. This phenomenon may explain the effect of water therapy performed in thermal spas, where the administration of 1-2 liters of mineral water is performed in 30-60 minutes. OBJECTIVES: The aim of the present study is to investigate if this increased transport capacity can act also in the renal tubular apparatus to modify the excretion of some endogenous substances. MATERIALS AND METHODS: We evaluated daily renal clearances in ten subjects under basal conditions during supplemental administration of 25 ml/kg of mineral water over a 24-hour period and during the administration of the same amount of water over a 30-minute period. RESULTS: Subjects who drank a water load of 25 ml/Kg over 30 minutes showed a higher diuresis than that observed in those who drank the same amount over a 24-hour period. Creatinine and urea clearance at 24 hours were significantly higher in subjects who drank the water load over 30 minutes. Serum magnesium levels and folic acid levels were also significantly higher in subjects who drank the water load over 30 minutes. CONCLUSIONS: Water administration over a short period of time seems to modify the daily excretion of some endogenous metabolites.

In vivo photoacoustic flow cytometry for monitoring of circulating single cancer cells and contrast agents.

A new photoacoustic flow cytometry was developed for real-time detection of circulating cells, nanoparticles, and contrast agents in vivo. Its capability, integrated with photothermal and optical clearing methods, was demonstrated using a near-infrared tunable laser to characterize the in vivo kinetics of Indocyanine Green alone and single cancer cells labeled with gold nanorods and Indocyanine Green in the vasculature of the mouse ear. In vivo applications are discussed, including selective nanophotothermolysis of metastatic squamous cells, label-free detection of melanoma cells, study of pharmokinetics, and immune response to apoptotic and necrotic cells, with potential translation to humans. The threshold sensitivity is estimated as one cancer cell in the background of 10(7) normal blood cells.