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Objectives To identify predictors and time trends over 10 years of psychiatric consultation or treatment in adolescents and young adults referring to Emergency Department (ED). Methods Real‐world cohort data from 50,056 adolescents and young adults referring 105,596 times to ED between 2007 and 2016. We tested whether gender, age, triage code (red, yellow, green, white with decreasing severity), and referral modality predicted primary (psychiatric consultation) or secondary outcomes (anxiolytic treatment, sedative treatment, psychiatric admission), and whether these outcomes increased over the last 10 years. Results Mean age was 19.57(SD = 2.52), female percentage was 48.77%. Overall, 6.93% underwent psychiatric consultation, treatment, or admission. Among 2,547 adolescents and young adults undergoing a psychiatric consultation, 58.07% had either yellow or red triage code, and 47.2% were brought by ambulance. Female gender predicted psychiatric consultation and anxiolytic treatment, male gender predicted sedative treatment, suggesting gender differences in help‐seeking behaviors. Older age predicted all outcomes. Severe triage presentation and being brought by ambulance increased the risk of primary and secondary outcomes. Psychiatric consultation (1.77% to 3.64%), anxiolytic (3.04% to 6.15%), or admission (0.40% to 0.98%) roughly doubled, and sedative (0.27% to 1.23%) treatment had a four‐fold increase from 2007 to 2016. Conclusions Among adolescents and young adults aged 15 to 24 years old ED appears to be necessary for young help‐seeking subjects given the severe presentations and the increasing number of adolescents referring to ED. More studies should assess whether ED might be helpful in detecting subjects with sub‐threshold or early psychiatric symptoms, or at risk for severe mental illness.

Several research groups have observed magnetism in monolayer-protected gold cluster samples, but the results were often contradictory, and thus, a clear understanding of this phenomenon is still missing. We used Au25(SCH2CH2Ph)180, which is a paramagnetic cluster that can be prepared with atomic precision and whose structure is known precisely. Previous magnetometry studies only detected paramagnetism. We used samples representing a range of crystallographic orders and studied their magnetic behaviors using electron paramagnetic resonance (EPR). As a film, Au25(SCH2CH2Ph)180 exhibits a paramagnetic behavior, but at low temperature, ferromagnetic interactions are detectable. One or few single crystals undergo physical reorientation with the applied field and exhibit ferromagnetism, as detected through hysteresis experiments. A large collection of microcrystals is magnetic even at room temperature and shows distinct paramagnetic, superparamagnetic, and ferromagnetic behaviors. Simulation of the EPR spectra shows that both spin-orbit (SO) coupling and crystal distortion are important to determine the observed magnetic behaviors. Density functional theory calculations carried out on single cluster and periodic models predict the values of SO coupling and crystal-splitting effects in agreement with the EPR-derived quantities. Magnetism in gold nanoclusters is thus demonstrated to be the outcome of a very delicate balance of factors. To obtain reproducible results, the samples must be (i) controlled for composition and thus be monodisperse with atomic precision, (ii) of known charge state, and (iii) well-defined in terms of crystallinity and experimental conditions.

Singlet oxygen, 1O2, is an excited oxygen species [1] that can be efficiently generated by photosensitizers. These are molecules that upon photoexcitation eventually generate a triplet excited state that can be quenched by the ground state oxygen triplet, 3O2, to form 1O2 [1]. The latter finds uses in many applications, especially organic synthesis, photocatalysis, and photodynamic therapy [2-4]. On the other hand, its possible use depends on its effective lifetime, as 1O2 is a very reactive species that decays via quenching by the solvent and/or interactions with other molecules in solution [1]. This is the case of the photosensitizers themselves, and this may result in a serious drawback for efficient applications of 1O2. In this area, the use of monolayer-protected metal nanoclusters as photosensitizers is limited. We carried out a systematic study on the photosensitizer behavior of a series of molecular Au24M(SR)18 clusters [5,6], where the R group and the doping metal M were varied. Electrochemical characterization allowed us to select the best R/M combinations to test a sufficiently large oxidation-potential range. 1O2 detection was carried out by time-resolved electron paramagnetic resonance [7], which allowed us to determine 1O2 lifetimes spanning more than one order of magnitude (on the microsecond time scale), depending on the specific cluster employed. Among the main results, we found that: (i) a more positive potential for the oxidation of the cluster is matched by a (beneficial) longer 1O2 lifetime; (ii) proper design of the electrochemical properties of the cluster yields the same results as those of reference photosensitizers, despite the unprecedented low oxidation potentials exhibited by these clusters; (iii) kinetic analysis based on the Marcus theory provides important insights into the mechanism of the quenching of 1O2 by gold nanoclusters, how to control it, and why properly doped gold nanoclusters may perform very well in 1O2 photosensitization [8]. Further effects, detected by electrochemistry, are under investigation. References [1] Bregnhoj, M.; Westberg, M.; Minaev, B. F.; Ogilby, P. R. Acc. Chem. Res. 2017, 50, 1920. [2] Turro, N. J.; Ramamurthy, V.; Scaiano, J. C. Molecular Oxygen and Organic Photochemistry. In Modern Molecular Photochemistry of Organic Molecules; University Science Books: Sausalito, CA, 2010; Ch. 14. [3] Ghogare, A. A.; Greer, A. Chem. Rev. 2016, 116, 9994. [4] Nosaka, Y.; Nosaka, A. Y. Chem. Rev. 2017, 117, 11302. [5] Antonello, S.; Maran, F. Curr. Opin. Electrochem. 2017, 2, 18. [6] Fei, W.; Antonello, S.; Dainese, T.; Dolmella, A.; Lahtinen, M.; Rissanen, K.; Venzo, A.; Maran, F. J. Am. Chem. Soc. 2019, 141, 16033. [7] Agrachev, M.; Ruzzi, M.; Venzo, A.; Maran, F. Acc. Chem. Res. 2019, 52, 44. [8] Agrachev, M.; Fei, W.; Antonello, S.; Bonacchi, S.; Dainese, T.; Zoleo, A.; Ruzzi, M.; Maran, F. Chem. Sci. 2020, 11, 3427.

Non-carbonate clay samples, fired in steps of 50 °C between 600 °C and 1000 °C, were examined through electron paramagnetic resonance (EPR) techniques (X- and Q-Band pulsed- and cw-EPR). X-band pulsed EPR and Q-Band pulsed- and cw-EPR techniques were able to spot 55 Mn(II) signals out of the large iron background in the full range of firing temperatures, allowing one to follow the phase transformations through the evolution of the manganese signal. Q-Band pulsed EPR turned out to be particularly suitable to detect even small amounts of this ion in calcite, while the 55 Mn hyperfine coupling constant, determined through Q-Band cw-EPR, could be used to detect transitions to the glassy phase.

A basic recipe for making a Cr logwood ink dating to 1847 was adapted in order to obtain hues varying from orange to dark brown. The inks synthesized were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, liquid chromatography–diode array detection–quadrupole time-of-flight–mass spectrometry, and liquid chromatography–inductively coupled plasma–mass spectrometry to gain insight into the spectroscopic properties of the compounds responsible for the inks colors and to obtain Raman spectra that can be used for their non-invasive identification in works of art and in historic documents. It was observed that hematein and logwood oxidation products are present in the inks and that their relative proportions determine the inks hues. It was also found that relatively small variations in the basic recipe, such as in the temperature that the logwood and K2CrO4 mixture were heated at, gave inks with different hues. The Raman spectra acquired in the laboratory samples were used to characterize the ink used by Vincent van Gogh in Street in Saintes-Maries-de-la-Mer. Copyright © 2016 John Wiley & Sons, Ltd.

Driving under the influence of alcohol and/or psychoactive substances increases the risk of road accidents, but it is controversial whether this affects site and severity of injuries.We search for alcohol, cannabinoids, cocaine, benzodiazepines, opioids, methadone, amphetamines and barbiturates in biological fluids of 1764 traumatized drivers admitted to the Emergency Department (ED) of Padua between 2010 and 2014.We note correlation between alcohol and benzodiazepines and admission in ICU and between all the intoxications and the reserved prognosis. The sites of injuries were: head (37.13%), maxillofacial (8.33%), spinal column (44.67%), thoracic (15.31%), abdominal (5%), pelvic (2.55%) and limb traumas (23.58%). We observed a correlation between head trauma (p0.0001), maxillofacial trauma (p = 0.0418), thoracic trauma (p = 0.0215), pelvic trauma (p = 0.0008), spinal column trauma (p0.0001) and the totality of the intoxication and an association between benzodiazepines and thoracic and pelvic trauma.Alcohol and benzodiazepines intoxication increases the risk of reserved prognosis and admission in ICU; benzodiazepines intoxication correlates with thoracic and pelvic trauma.

Scientific analyses of ancient glasses have been carried out for many years using elemental chemical analysis. However, it is known that the control of the redox conditions in the glass melt has a strong implication on the final hue of glass because it affects Fe2+/SFe. Therefore an increasing number of studies on the redox conditions have been published in recent years by means of synchrotron based Xray absorption spectroscopy. This is a technique which is not easily accessible and requires dedicated facilities. In this paper we describe an alternative approach by means of optical absorption spectroscopy. We synthesised 10 soda-lime-silica glasses with known redox conditions and iron concentration to calibrate the absorption at 1100 nm as a function of Fe2+ concentration. The linear extinction coefficient was also determined. These glasses were also studied by means of X-ray Absorption Near Edge Structure (XANES) spectroscopy. Electron paramagnetic resonance spectroscopy was additionally used as an ancillary method to verify the quality of our data. Furthermore 28 samples from real archaeological samples were analysed by XANES and optical spectroscopy as a case study. The Fe2+/SFe values obtained were compared and demonstrated that the two techniques were in good agreement with each other. Optical spectroscopy can be applied in situ with moderate sample preparation to determine the concentration of Fe2+. To investigate the redox conditions, especially as a first screening approach, this methodology is an important tool to take into consideration before applying more sophisticated techniques such as XANES, which is more elaborate and requires high-tech resources.

The monoanion of bis-TRANS2-[ N -methyl-3,4-fulleropyrrolidine] (TRANS2 − ) was investigated by continuous wave (CW) and pulsed EPR. X-band spectra showed signals due to 14 N and 13 C hyperfine couplings (hcc). DFT calculations of 14 N and 13 C isotropic hcc were performed on TRANS2 − for different conformations of the pyrrolidinic rings. Calculated values, averaged over the conformations, match well with the experimental values. Accurate g-tensor principal values were obtained by high frequency EPR. The possible dynamical processes affecting the electron spin relaxation are discussed to explain the temperature dependence of the EPR line widths and of the T 2 and T 1 relaxation times obtained by pulsed methods.

In this work we report a pre-restoration spectroscopic study by a multi-technique approach on the green pigment and the binder used in illuminations in a codex of the late 15th century. Our analysis aimed to characterize the badly deteriorated parts of the codex, mainly the green parts, by electron paramagnetic resonance (EPR), X-ray diffraction (XRD), X-ray fluorescence (XRF) and infrared spectroscopy (ATR-IR). We will show that a combined study by EPR and IR spectroscopies on model systems allowed us to refine the general information obtained with the other techniques and to identify the binding media surrounding the chromophore Cu(II) ion.

The radical anions of three C60 bisadducts (from now on termed CIS1, CIS2, and CIS3) have been studied in liquid solution and glassy matrix by X-band and high frequency CW-EPR spectroscopy. The three adducts CIS1, CIS2, and CIS3 are characterized by a pyrrolidinic ring and an isoxazolinic ring in position cis1-O, cis2-C, and cis3-C, respectively; the two rings are connected by a methylenic chain. In the X-band spectra of CIS1, one species has been observed showing hyperfine coupling constants with the pyrrolidinic nitrogen and View the MathML source nuclei, whereas in the X-band spectra of CIS2 and CIS3 more species are apparent, some with and other without hyperfine coupling with the pyrrolidinic View the MathML source. High field spectra at 110 and 220 GHz for all the bisadducts have been obtained; in all the cases, more species are evident with small differences in the g-factors. The occurrence of more species are discussed and put in relation with different bisadduct conformations. Furthermore, on the basis of the mechanism proposed for the View the MathML source hcc in previously studied fulleropyrrolidine (FP) monoanions, the presence or absence of View the MathML source hcc for CIS1 and the different conformations of CIS2 and CIS3 is discussed. The temperature dependence of the EPR linewidths and the g-factors of all the species have been determined and discussed in term of the different structural stiffness and symmetry of the three bisadducts.

We prepared four structurally related paramagnetic Au25(SR)018 clusters. 1H ENDOR and NMR signals could be successfully related to the types of ligand and the distance of protons from the central gold core. It is shown that the unpaired electron can be used as a very precise probe of the structure of the interface between metal core and capping ligands., The field of molecular metal clusters protected by organothiolates is experiencing a very rapid growth. So far, however, a clear understanding of the fine interactions between the cluster core and the capping monolayer has remained elusive, despite the importance of the latter in interfacing the former to the surrounding medium. Here, we describe a very sensitive methodology that enables comprehensive assessment of these interactions. Pulse electron nuclear double resonance (ENDOR) was employed to study the interaction of the unpaired electron with the protons of the alkanethiolate ligands in four structurally related paramagnetic Au25(SR)018 clusters (R = ethyl, propyl, butyl, 2-methylpropyl). Whereas some of these structures were known, we present the first structural description of the highly symmetric Au25(SPr)018 cluster. Through knowledge of the structural data, the ENDOR signals could be successfully related to the types of ligand and the distance of the relevant protons from the central gold core. We found that orbital distribution affects atoms that can be as far as 6 Å from the icosahedral core. Simulations of the spectra provided the values of the hyperfine coupling constants. The resulting information was compared with that provided by 1H NMR spectroscopy, and molecular dynamics calculations provided useful hints to understanding differences between the ENDOR and NMR results. It is shown that the unpaired electron can be used as a very precise probe of the main structural features of the interface between the metal core and the capping ligands.

An endofulleropyrrolidine, with H2 as a guest, has been reduced to a paramagnetic endofulleride radical anion. The magnetic interaction between the electron delocalized on the fullerene cage and the guest H2 has been probed by pulsed ENDOR. The experimental hyperfine couplings between the electron and the H2 guest were measured, and their values agree very well with DFT calculations. This agreement provides clear evidence of magnetic communication between the electron density of the fullerene host cage and H2 guest. The ortho-H2/para-H2 interconversion is revealed by temperature-dependent ENDOR measurements at low temperature. The conversion of the paramagnetic ortho-H2 to the diamagnetic para-H2 causes the ENDOR signal to decrease as the temperature is lowered due to the spin catalysis by the paramagnetic fullerene cage of the radical anion fulleride.

Samples from covers and endleaves of sixteenth to eighteenth century books in good conservation state from the Biblioteca Nazionale Marciana in Venice have been investigated by means of electron paramagnetic resonance (EPR) spectroscopy and optical microscopy to characterize the embedded paramagnetic species and to test differences and similarities of the materials in the two types of book components. We detected the Mn(II), Fe(III), and Cu(II) paramagnetic ions, and analyzed their EPR signals by comparing them with previous results in literature of EPR studies on paper. Interestingly, the Mn(II) EPR spectrum profile appears as a fingerprint for samples coming from the same book, either from cover or endleaf, and it looks different for samples extracted from different books. We discuss the role of EPR as a spectroscopic tool for characterizing the interaction of the ions with the paper components and as possible agents of degradation.

The cross-linked polymer EnCat™ 40 (polyurea) is investigated on the micro-(ESEM) and nanometer scale (ISEC, ESR, 13 C CP MAS NMR) both in the dry (ESEM, 13 C CP MAS NMR) and in the swollen state (ISEC, ESR, 13 C CP MAS NMR). After swelling with THF or DMF, nanoporous domains featured by ca. 1.2 nm pores are clearly detected with ISEC. ESR analysis based on the evaluation of the rotational mobility of a suitable spin probe dissolved in THF, DMF, MeOH and PhCH 3 as swelling media, does qualitatively confirm the presence of nanoporosity also in MeOH- and PhCH 3 -swollen EnCat™ 40. Our manifold analysis reveals the lack of any detectable nanoporosity in water.

Electron Paramagnetic Resonance (EPR) is a technique devoted to the identification and characterization of paramagnetic species, i.e. chemical species with unpaired electrons. Very common paramagnetic species which can be detected through EPR in historic paper are Fe(III), Mn(II), Cu(II) ions and radicals, where Fe(III), Cu(II) and radicals play a relevant role in paper degradation. Specifically, Fe(III) is almost ubiquitous in historic paper. Here we propose an overview of the EPR signals in historic and artificially aged paper, and in particular, we would like to show how a deep analysis of EPR signals from paper could provide useful information about the paper’s origin and unique indications of the degradation and oxidation level of the paper.

The high-affinity metal-binding site of isolated F(1)-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F(1) was fully depleted of endogenous Mg(II) and nucleotides [stripped F(1) or MF1(0,0)] and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP). Mn(II) and nucleotides were added to MF1(0,0) either subsequently or together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that in all samples Mn(II) enters one catalytic site on a beta subunit. From the HF-EPR spectra, the zero-field splitting (ZFS) parameters of the various samples were obtained, showing that different metal-protein coordination symmetry is induced depending on the metal nucleotide addition order and the protein/metal/nucleotide molar ratios. The electron spin-echo envelope modulation (ESEEM) technique was used to obtain information on the interaction between Mn(II) and the (31)P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured (31)P hyperfine couplings clearly indicated coordination changes related to the metal nucleotide addition order and the protein/metal/nucleotide ratios. On the contrary, the samples with AMPPNP showed very similar ESEEM patterns, despite the remarkable differences present among their HF-EPR spectra. This fact has been attributed to changes in the metal-site coordination symmetry because of ligands not involving phosphate groups. The kinetic data showed that the divalent metal always induces in the catalytic site the high-affinity conformation, while EPR experiments in frozen solutions supported the occurrence of different precatalytic states when the metal and ADP are added to the protein sequentially or together as a preformed complex. The different states evolve to the same conformation, the metal(II)-ADP inhibited form, upon induction of the trisite catalytic activity. All our spectroscopic and kinetic data point to the active role of the divalent cation in creating a competent catalytic site upon binding to MF1, in accordance with previous evidence obtained for Escherichia coli and chloroplast F(1).

X-ray crystallography has been fundamental in discovering fine structural features of ultrasmall gold clusters capped by thiolated ligands. For still unknown structures, however, new tools capable of providing relevant structural information are sought. We prepared a 25-gold atom nanocluster protected by the smallest ligand ever used, ethanethiol. This cluster displays the electrochemistry, mass spectrometry, and UV-vis absorption spectroscopy features of similar Au25 clusters protected by 18 thiolated ligands. The anionic and the neutral form of Au25(SEt)18 were fully characterized by (1)H and (13)C NMR spectroscopy, which confirmed the monolayer's properties and the paramagnetism of neutral Au25(SEt)18(0). X-ray crystallography analysis of the latter provided the first known structure of a gold cluster protected by a simple, linear alkanethiolate. Here, we also report the direct observation by electron nuclear double resonance (ENDOR) of hyperfine interactions between a surface-delocalized unpaired electron and the gold atoms of a nanocluster. The advantages of knowing the exact molecular structure and having used such a small ligand allowed us to compare the experimental values of hyperfine couplings with DFT calculations unaffected by structure's approximations or omissions.

Background —The purpose of this study was to investigate whether some cellular and molecular features of tissue retrieved at carotid endarterectomy are associated with the extent of neointima formation at ultrasound follow-up. Methods and Results —One hundred fifty patients were studied. Endarterectomy specimens were tested by immunocytochemistry with the use of (1) monoclonal antibodies that identify smooth muscle cells (SMCs) and fetal-type SMCs on the basis of smooth muscle and nonmuscle myosin content, (2) the anti-macrophage HAM 56, and (3) the anti-lymphocyte CD45RO. The maximum intima-media thickness (M-IMT) of the revascularized vessel was assessed by the use of B-mode ultrasonography 6 months after surgery. The M-IMT values were related positively to the number of SMCs ( r =0.534, P r =−0.428, P P =0.001, respectively). Patients were classified as class 1 (M-IMT ≤1.0 mm), class 2 (1.01.3 mm). An abundance of SMCs, mostly of fetal type, was found in the plaque of class 3 patients, whereas lesions from class 1 patients were rich in macrophages and lymphocytes. In the multivariate analysis, factors related to M-IMT were the number of SMCs and the percentage of fetal-type SMCs present in the plaque. Conclusions —Although the classic risk factors did not play a role, an abundance of SMCs and a scarcity of macrophages characterized the primary lesion of patients in whom neointima developed after surgery. In patients in whom neointima did not develop, lesions were rich in macrophages and lymphocytes. This approach can be useful in defining patients at risk of restenosis.

The radical anions of three C60 derivatives (N-methyl-3,4-fulleropyrrolidine, N-methyl-2-(3,6,9-trioxadecyl)3,4-fulleropyrrolidine, and bis-N-methyl-3,4-fulleropyrrolidine are respectively MFP - , tegMFP - , and bisMFP - ) have been investigated by continuous wave (cw) and pulsed EPR in methyltetrahydrofuran and tetrahydrofuran solutions. The EPR spectra for MFP - and tegMFP - show a 1:1:1 triplet, due to the hyperfine coupling with the pyrrolidine nitrogen. The EPR spectrum of bisMFP - shows a superimposition of two signals, each due to a 1:2:3:2:1 quintet. Each quintet is due to the hyperfine coupling with the two pyrrolidine nitrogen atoms. The two signals are attributed to two different conformers of the radical. A direct transfer of spin density from the C60 sphere to the nitrogen atoms is discussed as arising from the bent conformation of the pyrrolidine rings. All the spectra show several weaker satellite lines, due to the radical anions containing a 13 C atom. From their analysis a C 2V symmetry for the spin distribution is obtained in the case of MFP - and tegMFP - . The EPR line widths of these latter radicals at room temperature are larger than those expected on the basis of spin relaxation due to rotational diffusion in solution, and the line widths decrease upon decreasing the temperature. Pulsed EPR measurements of T1 in liquid and frozen solutions show that the EPR line widths are determined by the anomalously short lifetimes of the spin states. The temperature dependence of T1 obeys to the Arrhenius law, giving an activation energy of 7 kJ/mol. The latter results are compared with those obtained in the past for the lone C60 . They are attributed to the closeness of the electronic levels, due to the weak perturbation of the pyrrolidine ring on the symmetrical electronic distribution of C 60. On the other hand, the spin relaxation properties of bisMFP - are shown those expected for a nonsymmetrical radical. The comparison of cw- and pulsed-EPR spectra of MFP - in frozen solutions of MeTHF and THF indicates the presence of aggregates of interacting radicals in THF.

The polymer framework of water-swollen copolymers of N,N-dimethylacrylamide, acrylamido-2-methylpropanesulfonic acid, and ethylenedimethacrylate (nominal cross-linking degrees of 4, 8, and 20 mol %) is composed of highly expanded domains, with "pores" not less than 6 nm in diameter. When the 4% cross-linked copolymer (DAE 26-4) is swollen with a 10(-4) M solution of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) in water, MeOH, EtOH, or nBuOH, the molecules of the paramagnetic probe rotate rapidly (τ

Human α-Synuclein (aS), a 140 amino acid protein, is the main constituent of Lewy bodies, the cytoplasmatic deposits found in the brains of Parkinson's disease patients, where it is present in an aggregated, fibrillar form. Recent studies have shown that aS is a metal binding protein. Moreover, heavy metal ions, in particular divalent copper, accelerate the aggregation process of the protein. In this work, we investigated the high affinity binding mode of truncated aS (1-99) (aS99) with Cu(II), in a stoichiometric ratio, to elucidate the residues involved in the binding site and the role of copper ions in the protein oligomerization. We used Electron Paramagnetic Resonance spectroscopy on the Cu(II)-aS99 complex at pH 6.5, performing both multifrequency continuous wave experiments and pulsed experiments at X-band. The comparison of 9.5 and 95 GHz data showed that at this pH only one binding mode is present. To identify the nature of the ligands, we performed Electron Spin Echo Envelope Modulation, Hyperfine Sublevel Correlation Spectroscopy, and pulsed Davies Electron-Nuclear Double Resonance (Davies-ENDOR) experiments. We determined that the EPR parameters are typical of a type-II copper complex, in a slightly distorted square planar geometry. Combining the results from the different pulsed techniques, we obtained that the equatorial coordination is {N(Im), N(-), H(2)O, O}, where N(im) is the imino nitrogen of His50, N(-) a deprotonated amido backbone nitrogen that we attribute to His50, H(2)O an exchangeable water molecule, and O an unidentified oxygen ligand. Moreover, we propose that the free amino terminus (Met1) participates in the complex as an axial ligand. The MXAN analysis of the XAS k-edge absorption data allowed us to independently validate the structural features proposed on the basis of the magnetic parameters of the Cu(II)-aS99 complex and then to further refine the quality of the proposed structural model.

High-frequency electron paramagnetic resonance (EPR) and X-band electron-nuclear double resonance (ENDOR) spectroscopies were used to investigate the effect of gamma-irradiation on single crystals of L-tyrosine hydrochloride at room temperature. The oxidation product is the tyrosyl radical formed by hydrogen abstraction from the phenolic group; interestingly, on freshly irradiated crystals, two tyrosyl radicals were identified, characterized by slightly different magnetic parameters. In particular, one of the two radicals, with a gxx value of 2.00621, has its phenoxyl oxygen strongly hydrogen-bonded to one or more donors; to our knowledge, this is the lower gxx value reported for tyrosyl radicals. These two oxidation radicals are found to evolve very slowly to a third, single more stable radical conformation. To interpret the experimental data, a possible molecular scenario is presented, where the process of radical formation can be seen as a hydrogen atom transfer or a proton-coupled electron transfer. These processes seem to be controlled by the specific network of hydrogen-bond interactions present in the crystal. The results are discussed in relation to their relevance for the interpretation of EPR spectra of tyrosyl radicals in biological systems.

The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures.

Although vascular smooth muscle cells (SMCs) play a key role in the development of atherosclerotic lesions, they are not a homogeneous cell type. Myosin has been used as a marker of SMC differentiation in order to identify distinct SMC populations in the adult rabbit aorta. The medial layer of the normal adult aorta contains predominantly ‘adult’ type SMCs, which express smooth muscle (SM) myosin isoforms exclusively, and a minority of ‘immature’ type SMCs, which coexpress SM and nonmuscle (NM) myosin isoforms. The size of this latter SMC subpopulation, showing the ‘immature’ pattern of myosin isoform expression, increases markedly in the aortic media during experimental atherogenesis, and represents a major SMC type in the atherosclerotic plaque. The dihydropyridine derivative, nifedipine, has a marked effect on NM myosin expression and SMC differentiation in vitro. In vivo, short term administration of nifedipine resulted in the disappearance of ‘immature’ type SMCs from the aortic media of both normocholesterolaemic and hypercholesterolaemic adult rabbits. Moreover, in a model of atherosclerosis prevention, nifedipine significantly reduced the area of aortic intimal thickening and reduced the size of the ‘immature’ type SMC population both in the aortic media and intima of the hypercholesterolaemic rabbit.

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