Feasibility of blood testing combined with PET-CT to screen for cancer and guide intervention.

Cancer treatments are often more successful when the disease is detected early. We evaluated the feasibility and safety of multicancer blood testing coupled with positron emission tomography-computed tomography (PET-CT) imaging to detect cancer in a prospective, interventional study of 10,006 women not previously known to have cancer. Positive blood tests were independently confirmed by a diagnostic PET-CT, which also localized the cancer. Twenty-six cancers were detected by blood testing. Of these, 15 underwent PET-CT imaging and nine (60%) were surgically excised. Twenty-four additional cancers were detected by standard-of-care screening and 46 by neither approach. One percent of participants underwent PET-CT imaging based on false-positive blood tests, and 0.22% underwent a futile invasive diagnostic procedure. These data demonstrate that multicancer blood testing combined with PET-CT can be safely incorporated into routine clinical care, in some cases leading to surgery with intent to cure.

Immobilization of large, aliovalent cations in the small-pore zeolite K-natrolite by means of pressure.

High-pressure ion exchange of small-pore zeolite K-natrolite allows immobilization of nominally non-exchangeable aliovalent cations such as trivalent europium. A sample exchanged at 3.0(1) GPa and 250 °C contains about 4.7 Eu(III) ions per unit cell, which is equivalent to over 90 % of the K(+) cations being exchanged.

Telecare system for cardiac surgery patients: implementation and effectiveness.

OBJECTIVES: To manage a patient's blood pressure and recovery, and to reduce unnecessary hospital visits after heart surgery, we developed and established a telecare service. METHODS: We established and test-operated the system that enabled biometric data to be measured and monitored at home, and directed connections to the video consultation with monitoring personnel and medical staff when abnormal symptoms were detected. RESULTS: As a result of using the telecare service with patients discharged from the hospital after undergoing heart surgery, the patients were mostly satisfied with the service and use of the equipment, and some patients wanted to actually receive the service continuously along with a device which could be more easily used. CONCLUSIONS: Telecare services are greatly needed for patients discharged after heart surgery for a certain period of time. A model should be developed which provides devices necessary for each disease in package form and customizes the content and services in one package.

Mutant proteins as cancer-specific biomarkers.

Cancer biomarkers are currently the subject of intense research because of their potential utility for diagnosis, prognosis, and targeted therapy. In theory, the gene products resulting from somatic mutations are the ultimate protein biomarkers, being not simply associated with tumors but actually responsible for tumorigenesis. We show here that the altered protein products resulting from somatic mutations can be identified directly and quantified by mass spectrometry. The peptides expressed from normal and mutant alleles were detected by selected reaction monitoring (SRM) of their product ions using a triple-quadrupole mass spectrometer. As a prototypical example of this approach, we demonstrated that it is possible to quantify the number and fraction of mutant Ras protein present in cancer cell lines. There were an average of 1.3 million molecules of Ras protein per cell, and the ratio of mutant to normal Ras proteins ranged from 0.49 to 5.6. Similarly, we found that mutant Ras proteins could be detected and quantified in clinical specimens such as colorectal and pancreatic tumor tissues as well as in premalignant pancreatic cyst fluids. In addition to answering basic questions about the relative levels of genetically abnormal proteins in tumors, this approach could prove useful for diagnostic applications.

Spectroscopic and density functional theory studies of the blue-copper site in M121SeM and C112SeC azurin: Cu-Se versus Cu-S bonding.

S K-edge X-ray absorption, UV-vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII-thioether model complex shows that the 38% S character in the ground state wave function of the blue-copper (BC) sites solely reflects the Cu-SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu-SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV-vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin-orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 A. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.

Perturbations to the geometric and electronic structure of the CuA site: factors that influence delocalization and their contributions to electron transfer.

Using a combination of electronic spectroscopies and DFT calculations, the effect of pH perturbation on the geometric and electronic structure of the CuA site has been defined. Descriptions are developed for high pH (pH = 7) and low pH (pH = 4) forms of CuA azurin and its H120A mutant which address the discrepancies concerning the extent of delocalization indicated by multifrequency EPR and ENDOR data (J. Am. Chem. Soc. 2005, 127, 7274; Biophys. J. 2002, 82, 2758). Our resonance Raman and MCD spectra demonstrate that the low pH and H120A mutant forms are essentially identical and are the perturbed forms of the completely delocalized high pH CuA site. However, in going from high pH to low pH, a seven-line hyperfine coupling pattern associated with complete delocalization of the electron (S = 1/2) over two Cu coppers (I(Cu) = 3/2) changes into a four-line pattern reflecting apparent localization. DFT calculations show that the unpaired electron is delocalized in the low pH form and reveal that its four-line hyperfine pattern results from the large EPR spectral effects of approximately 1% 4s orbital contribution of one Cu to the ground-state spin wave function upon protonative loss of its His ligand. The contribution of the Cu-Cu interaction to electron delocalization in this low symmetry protein site is evaluated, and the possible functional significance of the pH-dependent transition in regulating proton-coupled electron transfer in cytochrome c oxidase is discussed.

A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures.

A temperature independent pH buffer has been developed from a combination of buffers of opposite-sign temperature coefficients, and utility in low temperature spectroscopy and storage of pH sensitive compounds is demonstrated.

Reorganization energy of the CuA center in purple azurin: impact of the mixed valence-to-trapped valence state transition.

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (approximately 0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.

Reduction potential tuning of the blue copper center in Pseudomonas aeruginosa azurin by the axial methionine as probed by unnatural amino acids.

The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.

Spectroscopic characterizations of bridging cysteine ligand variants of an engineered Cu2(Scys)2 CuA azurin.

Bridging cysteine ligands of the Cu(A) center in an engineered Cu(A) azurin were replaced with serine, and the variants (Cys116Ser and Cys112Ser Cu(A) azurin) were characterized by mass spectrometry, as well as UV-vis and electron paramagnetic resonance (EPR) spectroscopic techniques. The replacements resulted in dramatically perturbed spectroscopic properties, indicating that the cysteines play a critical role in maintaining the structural integrity of the Cu center. The replacements at different cysteine residues resulted in different perturbations, even though the two cysteines are geometrically symmetrical in the primary coordination sphere with respect to the two copper ions. The Cys112Ser variant contains two distinct type 2 copper centers, while the Cys116Ser variant has one type 1 copper center with slight tetragonal distortion. Both the UV-vis and EPR spectra of the Cys116Ser variant change with pH, and the pK(a) of the transition is 6.0. A type 1 copper EPR spectrum with A(||) = 26 G was obtained at pH 7.0, while a type 2 copper EPR spectrum with A(||) = 140 G was found at pH 5.0. Interestingly, lowering the temperature from 290 to 85 K resulted in conversion of the Cys116Ser variant from a type 1 copper center to a type 2 copper center, suggesting rearrangement of the ligand around the copper or binding of an exogenous ligand at low temperature. This difference in mutation effects at different cysteines may be due to different constraints exerted on the two cysteines by hydrogen-bonding patterns in the ligand loop.

Blue ferrocenium azurin: an organometalloprotein with tunable redox properties.

A ferrocene derivative (2-[(methylsulfonyl)thio]ethylferrocene) (1) has been synthesized and incorporated into apo-azurin from Pseudomonas aeruginosa by covalent attachment to the highly conserved Cys112. The resulting artificial organometalloprotein (a protein containing organometallic compounds in the active site) has been characterized by UV-vis, electrospray mass spectrometry, and cyclic voltammetry (CV). Incorporation of 1 into azurin resulted in a higher solubility of the ferrocene group and improved stability of the ferrocenium species in aqueous solution, as shown by a more intense UV-vis absorption and a more reversible CV of the attached ferrocene group, respectively. The incorporation of 1 also increased the reduction potential of the complex from 402 to 579 mV (vs NHE), consistent with the ferrocene group being encapsulated inside the hydrophobic environment of the protein. Modulation of the reduction potential of ferrocene by residues near the secondary coordination sphere has also been demonstrated. Raising the pH from 4 to 9 resulted in a greater than 80 mV decrease in reduction potential of the protein-bound ferrocene (from 579 to 495 mV), while replacing Met121, an amino acid residue in close proximity to the ferrocene group with a positively charged Arg or negatively charged Glu, resulted in the predicted increase or decrease in reduction potential at all pH values. Similarly, substitution of Met121 with a more hydrophobic Leu raised the reduction potential. The increased solubility, stability, and tune-ability of this organometalloprotein make it an ideal choice for carrying out a number of biological reactions, such as long-range electron transfer or sensing. As an example of such applications, stoichiometric oxidation of ferrocytochrome c by the blue ferrocenium azurin was demonstrated.

Axial methionine has much less influence on reduction potentials in a CuA center than in a blue copper center.

The role of the highly conserved axial methionine of the purple CuA center in an engineered CuA azurin on modulating the reduction potentials of the copper center was investigated by a systematic replacement of the methionine with glutamate, aspartate, and leucine. In contrast to the same substitutions in the structurally related blue copper azurin, much smaller changes in reduction potential were observed in the CuA azurin upon replacing the methionine ligand with negatively charged Glu (-8 mV) and Asp (-5 mV) and more hydrophobic Leu (+16 mV). These findings are important in understanding the different roles of the two cupredoxins. The diamond core Cu2S2(Cys) structure of the CuA is much more resistant to variations of axial ligand interactions than the distorted tetrahedral structure of the blue copper protein. This difference may translate into a much wider range of reduction potentials (>1000 mV) for blue copper proteins that transfer electrons to a variety of partners in many different biological systems and a much narrower range of reduction potentials (<40 mV) for CuA proteins where a small difference in reduction potentials between the CuA and its redox partners is required.

pH-dependent transition between delocalized and trapped valence states of a CuA center and its possible role in proton-coupled electron transfer.

A pH-dependent transition between delocalized and trapped mixed valence states of an engineered CuA center in azurin has been investigated by UV-visible absorption and electron paramagnetic resonance spectroscopic techniques. At pH 7.0, the CuA azurin displays a typical delocalized mixed valence dinuclear [Cu(1.5)....Cu(1.5)] spectra with optical absorptions at 485, 530, and 760 nm, and with a seven-line EPR hyperfine. Upon lowering of the pH from 7.0 to 4.0, the absorption at 760 nm shifted to lower energy toward 810 nm, and a four-line EPR hyperfine, typical of a trapped valence, was observed. The pH-dependent transition is reversible because increasing the pH restores all delocalized spectral features. Lowering the pH resulted in not only a trapped valence state, but also a dramatically increased reduction potential of the Cu center (from 160 mV to 340 mV). Mutation of the titratable residues around the metal-binding site ruled out Glu-114 and identified the C-terminal histidine ligand (His-120) as a site of protonation, because the His120Ala mutation abolished the above pH-dependent transition. The corresponding histidine in cytochrome c oxidases is along a major electron transfer pathway from CuA center to heme a. Because the protonation of this histidine can result in an increased reduction potential that will prevent electron flow from the CuA to heme a, the CuA and the histidine may play an important role in regulating proton-coupled electron transfer.

Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple CuA azurin.

Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence Cu(A) center in engineered Cu(A) azurin have been investigated by UV-visible (UV-vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the Cu(A) azurin resulted in a new absorption band around 500 nm in the UV-vis and an isotropic line at g = 2.16 in the EPR spectra. Control experiments, including additions of Cu(II)SO(4) or Cu(I)(CH(3)CN)(4)PF(6) to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide-Cu(A) azurin adduct indicate complex formation between cupric ion and ferrocyanide ion in the protein. Solvent or small molecule accessibility, metal oxidation state and the presence of more than one metal ion are potential factors important for the complex formation. These findings must be taken into consideration when using ferricyanide or ferrocyanide as redox agents for studying Cu(A) centers in proteins.

Determination of reduction potential of an engineered Cu(A) azurin by cyclic voltammetry and spectrochemical titrations.

The reduction potentials of an engineered CuA azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4'-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured CuA azurin are the same within the experimental error (422 +/- 5 and 425 +/- 5 mV vs. NHE, respectively, in 50 mM ammonium acetate buffer, pH 5.1, 300 mM NaCl, 25 degrees C), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured CuA azurin have been determined to be 271 +/- 7 mV (50 mM ammonium acetate buffer, pH 5.1, 4 degrees C) and 420 +/- 1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 degrees C), respectively. Spectroscopic redox titration using [Ru(NH3)5Py]2+ resulted in a reduction potential (254+/-4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 degrees C) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH3)5Py]2+-reduced CuA azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH3)5Py]2+ measures the reduction potential of native CuA azurin.