The role of laboratory medicine in a value-based healthcare system: the example of heart failure patient management in the Italian context.

OBJECTIVE: As of today, healthcare systems worldwide face severe challenges that undermine their sustainability. The value-based healthcare (VBHC) approach has been proposed as a strategic and methodological framework to ensure the delivery of the best patient outcomes with economic efficiency. Through the illustrative example of B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) for heart failure (HF) patient management in the context of the Italian National Healthcare system, this article explores the role that in vitro diagnostics (IVDs) can play in enabling value-based care models. SUBJECTS AND METHODS: 14 healthcare professionals representing the relevant professional figures involved in HF patient management met to revise the current HF patient journey and design a new care pathway that, leveraging on BNP/NT-proBNP, reflects the VBHC principles. RESULTS: The literature recognizes the dosage of BNP/NT-proBNP as the gold stan-dard for diagnosing HF. However, as of today, these IVDs are not employed at their full potential regarding HF patient management. A new patient journey is proposed so that patients are diagnosed early and properly monitored in the aftermath of hospitalization, improving outcomes at contained costs. CONCLUSIONS: As testified by the example of HF patient management in Italy, laboratory medicine can represent a lever for adopting value-based care models. Still, large-scale adoption of VBHC will call for structural reforms that revise how healthcare delivery is organized, measured, and reimbursed.

Methods for Solving Highly Symmetric De Novo Designed Metalloproteins: Crystallographic Examination of a Novel Three-Stranded Coiled-Coil Structure Containing d-Amino Acids.

The core objective of de novo metalloprotein design is to define metal-protein relationships that control the structure and function of metal centers by using simplified proteins. An essential requirement to achieve this goal is to obtain high resolution structural data using either NMR or crystallographic studies in order to evaluate successful design. X-ray crystal structures have proven that a four heptad repeat scaffold contained in the three-stranded coiled coil (3SCC), called CoilSer (CS), provides an excellent motif for modeling a three Cys binding environment capable of chelating metals into geometries that resemble heavy metal sites in metalloregulatory systems. However, new generations of more complicated designs that feature, for example, a d-amino acid or multiple metal ligand sites in the helical sequence require a more stable construct. In doing so, an extra heptad was introduced into the original CS sequence, yielding a GRAND-CoilSer (GRAND-CS) to retain the 3SCC folding. An apo-(GRAND-CSL12DLL16C)3 crystal structure, designed for Cd(II)S3 complexation, proved to be a well-folded parallel 3SCC. Because this structure is novel, protocols for crystallization, structural determination, and refinements of the apo-(GRAND-CSL12DLL16C)3 are described. This report should be generally useful for future crystallographic studies of related coiled-coil designs.

Ferric ion sequestering agents: kinetics of iron release from ferritin to catechoylamides.

The removal of ferric ion from the iron storage protein ferritin to synthetic catechoylamide sequestering agents has been studied using visible spectroscopy at 487 nm. One ligand which has been investigated in detail is N,N',N' ',-tris(2,3-dihydroxy-5-sulfobenzoyl)-1,5,10-triazadecane (3,4-LICAMS), which octahedrally coordinates the metal ion via six phenolic oxygens. For some related catechoylamide chelates, the percentage of iron removed after 6 h has been determined. These ligands incorporate various modifications, either on the catechol moiety or on the backbone structure of the ligand. Mobilization of iron by the catechoylamide ligands alone results in very slow exchange, and virtually no iron removal after 6 h. In contrast, addition of ascorbic acid to the reaction mixture facilitates iron exchange, with the release of 7% of the available iron in the same time span. Variation of the initial rate with ascorbic acid concentration results in Michaelis-Menten kinetics with Km = 1.7 . 10(-3) M and a maximal rate of 1.28 . 10(-7) M . min-1. The ascorbic acid-mediated rate was not affected by changing the catechoylamide ligand concentration, and was only slightly affected by variation of the ligand employed. These data are consistent with a multistep process which includes diffusion of a reductant into the ferritin inner core, reduction and possible chelation of the ferrous ion, diffusion out of the protein shell, and subsequent iron exchange with the catechoylamide molecule.

Recent advances in the understanding of the biological chemistry of manganese.

Developments in manganese biochemistry have centered on the discovery of new manganese enzymes, X-ray analysis of binuclear manganese enzymes, and the discovery of new spectroscopic signatures for the oxygen-evolving complex. Despite these gains, many questions regarding the structure, composition and redox state of the oxygen-evolving complex remain unanswered.

The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils.

To define the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded alpha-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pKa of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.

Fluconazole susceptibility of Italian Candida dubliniensis clinical isolates determined by reference and simplified tests.

Candida dubliniensis ia an opportunistic pathogen mainly associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. We recently recovered the first Italian clinical isolates of C. dubliniensis from the oral cavities of seven HIV-seropositive subjects. The in vitro susceptibility to fluconazole (FLCZ) of these isolates was determined according to the National Committee for Clinical Laboratory Standards (NCCLS) M27-A broth microdilution method for yeasts. All seven isolates of C. dubliniensis were susceptible to FLCZ (MICs < or =0.5 microg/ml). Results of this reference method were compared to those obtained with simplified tests, more adapted to routine evaluation in hospital laboratories. Fungitest and Sensititre YeastOne colorimetric microplate-based methods have been evaluated. The agar disk diffusion method has also been tested on two different media: RPMI 1640-2% glucose and High Resolution-2% glucose-0.5 microg/ml methylene blue. All of the simplified methods tested were able to correctly identify FLCZ-susceptibility of this group of Italian C. dubliniensis isolates.

[Anal manifestations of lichen planus]. / Manifestaciones anales del liquen plano.

Lichen planus (LP) is an entity endowed with a striking polimorphysm and with a diversity of topographic localizations. In spite of the diagnostic difficulties that the combination of these factors occasionally produce, the histopathology of the lesion is tipical and diagnostic. Our presentation seems to be justified since, to the best of our knowledge, the anal localization of the disease has not been reported and it is also rare. The finding of these anal manifestation has not been casual. With the certitude that there was no reason why LP could not appear in a semimucosa so much alike that of the lips we have systematically examined the anal region of patients bearing lesions of LP elsewhere. Anal lesions that appeared to correspond to LP were detected and their histopathology confirmed the existence of LP in some of these cases. The structural changes permit the grouping of cases in four categories: Tipical LP of the anal semimucosa. LP of the anal semimucosa masked by coexistent lichen simplex chronicus (neurodermatitis). Serial sections of the paraffin block or biopsy of a nearby area usually prove diagnostic. Neurodermatitis associated to a lichenoid tissue reaction: Focal effacement of the basal membrane associated to lymphoid infiltrate of the upper dermis. Neurodermatitis without lichenoid features. It is our interpretation that this localization of LP may be due to a Köbner's phenomenon provoked by scratching induced by pruritus, as it occurred in our cases, or by other microtraumas so common to the area.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cryptococcosis. Apropos of a case]. / Criptococcosis. A propósito de una observación.

A case of cryptococcosis in a woman 46 years old, with lesions of the skin and the lung with an evolution of six years who answered satisfactory to a treatment with ketoconazole. We will discuss the duration of the lesion, the possible start of the skin aspect of the disease and the excellent response obtained with ketoconazole.

[Benign circinated pityriasic erythema in children]. / Eritema circinado pitiriásico benigno del niño.

The case is report of a boy aged 6 months whose dermatosis had set in suddenly 15 days before in the form of large round circinate patches on the face, thorax, abdomen, dorsum and extremities. These patches were limited by a slightly elevated erythematous border, the inside of which was constituted by collarette-like epidermal desquamations with central parakerotosis. The patches impairment was observed in the child's general condition. A histopathological study was carried out.

Mechanism for the reduction of the mixed-valent MnIIIMnIV[2-OHsalpn]2+ complex by tertiary amines.

The mixed-valent dimanganese(III/IV) complex MnIIIMnIV(2-OHsalpn)2+, 1, is cleanly reduced in acetonitrile by aliphatic tertiary amines to give the dimanganese(III) product MnIII2(2-OHsalpn)2, 2. Thorough characterization of the organic reaction products shows that tributylamine is converted to dibutylformamide and propionaldehyde. Kinetic studies and radical trapping experiments suggest that this occurs via initial single-electron transfer from the amine to 1 coupled with C-H alpha proton transfer from the oxidized amine. EPR spectroscopy and base inhibition studies indicate that coordination of the amine to 1 is a critical step prior to the electron transfer step. Rate data and its dependence on the amine indicate that the ability of the amine to reduce 1 is correlated to its basicity rather than to its reduction potential. Weakly basic amines were unable to reduce 1 irrespective of their reduction potential. This was inferred to indicate that proton transfer from the amine radical cation is also important in the reduction of 1 by tertiary amines. Comparison of the activation energy with reaction thermodynamics indicates that proton transfer and electron transfer must be concerted to explain the rapidity of the reaction. The fate of the amine radical is dependent on the presence of oxygen, and labeling studies show that oxygen in the organic products arises from dioxygen, although incorporation from trace water was also observed. These data indicate that inhibition of the hydrolytic quenching of the amine radical in an aprotic solvent results in a different fate for the amine radical when compared to amine oxidation reactions in aqueous solution. The proposed mechanism gives new insight into the ability of amines with high reduction potential to reduce metal ions of lower potential. In particular, these data are consistent with the ability of small amines and certain amine-containing buffers to inhibit manganese-dependent oxygen evolution in photosynthesis, which arises in some cases as a result of manganese reduction and its concomitant loss from the PS II reaction center.

Thermodynamic binding constants for gallium transferrin.

Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well established that Ga3+ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO3 have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO3 are log K1 = 20.3 and log K2 = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe3+ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log [Ga-(H2O)6]) to express the effective binding strength at pH 7.4.

Copper-induced expression, cloning, and regulatory studies of the plastocyanin gene from the cyanobacterium Synechocystis sp. PCC 6803.

Plastocyanin can be detected in Synechocystis sp. PCC 6803 when 3 microM copper is added to the growth medium, BG-11. The plastocyanin gene (petE) was cloned from a genomic lambda EMBL 3 library by screening with the petE gene from Anabaena sp. PCC 7937. The Synechocystis 6803 petE gene is present as a single copy and, as deduced from the DNA sequence, encodes a precursor protein of 126 amino acids. The predicted 29 amino acid transit peptide shows substantial homology to the Anabaena 7937 transit peptide, thought to direct the plastocyanin precursor to the thylakoid lumen. Putative promoter sites -16 and -38 base pairs from the start of the petE gene have been identified. The deduced amino acid sequence has the greatest homology (61%) to the green alga Scenedemus obliquus plastocyanin. Despite the lower homology, the copper binding residues and certain aromatic residues remain highly conserved. Northern hybridization analysis indicates that the Synechocystis sp. PCC 6803 petE gene is not transcriptionally regulated since the accumulation of petE mRNA appears to be independent of the copper concentration in the growth media. The possibility of an additional polypeptide needed to facilitate the electron transfer from plastocyanin to P700+ is also discussed.

The effect of protonation on [Mn(IV)(µ2-O)] 2 complexes.

The series of complexes [Mn(IV)(X-SALPN)(µ2-O)]2, 1: X=5-OCH3; 2: X=H; 3: X=5-Cl; 4: X=3,5-diCl; 5: X=5-NO2, contain [Mn2O2](4+) cores with Mn-Mn separations of 2.7 Å. These molecules can be protonated to form [Mn(IV)(X-SALPN)(µ2-O,OH)]2 (+) in which a bridging oxide is protonated. The pKa values for the series of [Mn(IV)(X-SALPN)(µ2-O,OH)]2 (+) track linearly versus the shift in redox potential with a slope of 84 mV/pKa. This observation suggests that the [Mn2O2](4+) core can be considered as a unit in which the free energy of protonation is directly related to the ability to reduce the Mn(IV) ion. The marked sensitivity of the reduction potential to the presence of protons presents a mechanism in which an enzyme can control the oxidizing capacity of an oxo manganese cluster by the degree and timing of oxo bridge protonation.

Structural characterization of the manganese(IV) Schiff-base complex MnIV(5-Cl-SALAHP)2.

Bis[3-(5-chlorosalicylideneamino)propanolato-O,N,O']manganes e(IV) methanol solvate, [Mn(C10H10ClNO2)2].CH3OH, Mr = 510.3, monoclinic, P2(1)/c, a = 11.949 (2), b = 7.530 (2), c = 25.777 (6) A, beta = 105.75 (2) degrees, V = 2232.4 (8) A3, Z = 4, Dx = 1.518 g cm-3, lambda(Mo K alpha) = 0.7107 A, mu = 7.98 cm-1, F(000) = 1502, T = 300 K, R = 0.0343, wR = 0.032 for 2113 unique reflections with (I) greater than 3 sigma(I). The title complex MnIV(5-Cl-SALAHP)2 [5-Cl-SALAHP = 3-(5-chlorosalicylideneamino)propanolato] displays a regular octahedral geometry. The 5-Cl-SALAHP ligand is tridentate, forming a meridional chelate with one phenolato oxygen (Mn-Oavg = 1.90 A), one alkoxide oxygen (Mn-Oavg = 1.85 A) and one imine nitrogen (Mn-Navg = 2.02 A) coordinated to the metal. Important angles described by the six atoms bound to manganese are all very close to either 180 or 90 degrees except the N-Mn-N angle which is 174.7 degrees. Previous studies have shown that MnIV(5-Cl-SALAHP)2 displays a rhombic EPR spectrum with well-resolved 55Mn hyperfine structure on gx, gy and gz. In contrast, Mn(SALADHP)2 [SALADHP = 2-methyl-2-(salicylideneamino)-1,3-propanediolato] shows a broad, ill-defined signal at g = 5.15 and a weak g = 2 component. The different spectral forms result from the extent of distortion of the MnIV octahedron. The reported structure is of potential importance to the understanding of the photosynthetic water-oxidizing system.

Determination of the screw sense specificity of bovine liver fructokinase.

Fructokinase from beef liver showed a clear reversal in specificity when the two isomers of ATP beta S were used as substrates with Mg2+ and Cd2+, with the Sp isomer having the higher V/K value with Mg2+ and the Rp isomer the higher value with Cd2+. The delta isomer of MgATP is thus the active form of the substrate. The substitution of sulfur for oxygen in the noncoordinated position of the beta-phosphate caused a 102-fold decrease in V/K over the value seen with MgATP, while substitution in the coordinated position gave a 21-fold decrease over the V/K value seen with CdATP. The Km values were little affected by sulfur substitution, showing that the wrong screw sense isomers were nonproductively bound almost as well as the correct ones. When ADP alpha S was used as a substrate in the reverse reaction, the Sp isomer showed the highest V/K value with both Mg2+ and Cd2+, suggesting that the metal ion is not coordinated to the alpha-phosphate during transphosphorylation. The failure of CrATP to act as a substrate for fructokinase suggests that the enzyme inserts one of its side chains into the inner coordination sphere of the metal ion during the reaction.