Experimental testing of a prototype cantilevered liquid-nitrogen-cooled silicon mirror.

This report presents testing of a prototype cantilevered liquid-nitrogen-cooled silicon mirror. This mirror was designed to be the first mirror for the new soft X-ray beamlines to be built as part of the Advanced Light Source Upgrade. Test activities focused on fracture, heat transfer, modal response and distortion, and indicated that the mirror functions as intended.

Experimental demonstration of a soft x-ray self-seeded free-electron laser.

The Linac Coherent Light Source has added a self-seeding capability to the soft x-ray range using a grating monochromator system. We report the demonstration of soft x-ray self-seeding with a measured resolving power of 2000-5000, wavelength stability of 10(-4), and an increase in peak brightness by a factor of 2-5 across the photon energy range of 500-1000 eV. By avoiding the need for a monochromator at the experimental station, the self-seeded beam can deliver as much as 50-fold higher brightness to users.

Narrow band imaging and high definition television in the endoscopic evaluation of upper aero-digestive tract cancer.

Narrow band imaging and high definition television are recent innovations in upper aero-digestive tract endoscopy. Aim of this prospective, non-randomized, unblinded study was to establish the diagnostic advantage of these procedures in the evaluation of squamous cell cancer arising from various upper aero-digestive tract sites. Between April 2007 and January 2010, 444 patients affected by upper aero-digestive tract squamous cell cancer, or previously treated for it, were evaluated by white light and narrow band imaging ± high definition television endoscopy, both in the pre-/intra-operative setting and during follow-up. Tumour resection was performed taking into account narrow band imaging and high definition television information to obtain histopathologic confirmation of their validity. Endoscopic and pathologic data were subsequently matched to obtain sensitivity, specificity, positive, negative predictive values, and accuracy. Overall, 110 (25%) patients showed adjunctive findings by narrow band imaging ± high definition television when compared to standard white light endoscopy. Of these patients, 98 (89%) received histopatological confirmation. The sensitivity, specificity, positive, negative predictive values, and accuracy for white light-high definition television were 41%, 92%, 87%, 82%, and 67%, for narrow band imaging alone 75%, 87%, 87%, 74%, and 80%, and for narrow band imaging-high definition television 97%, 84%, 88%, 96%, and 92%. The highest diagnostic gain was observed in the oral cavity and oropharynx (25%). Narrow band imaging and high definition television were of value in the definition of superficial tumour extension, and in the detection of synchronous lesions in the pre-/intra-operative settings. These technologies also played an important role during post-treatment surveillance for early detection of persistences, recurrences, and metachronous tumours.

Endoscopic treatment of Zenker's diverticulum by carbon dioxide laser.

The management of Zenker's diverticulum remains controversial, as both external and endoscopic procedures are potentially associated with post-operative complications and risks. The endoscopic approach is based on cricopharyngeal myotomy or myectomy by laser, cautery or stapling. A retrospective chart review has been made from December 1994 to April 2009 of all patients with Zenker's diverticulum treated by endoscopic cricopharyngeal myectomy using CO(2) laser at the Department of Otorhinolaryngology - Head and Neck Surgery of the University of Brescia, Italy. Of the 28 patients (19 males, 9 females; mean age, 64 years; range, 46-95) scheduled for the trans-oral procedure, 27 (96%) were endoscopically treated. Diagnosis was based on the patient's history, flexible fiberoptic examination of the larynx, hypopharynx, and oesophagus, and videofluoroscopy with barium. Exclusion criteria included severe medical co-morbidities, impossibility to endoscopically expose the diverticulum, and small (< 2 cm) or large (> 6 cm) diverticula. The endoscopic procedure was performed using a CO(2) laser to section the cricopharyngeal muscle and remove the posterior part in order to obtain partial myectomy. Major complications occurred in 2 (7%) patients. No cases of recurrent nerve paralysis, pharyngo-cutaneous fistula, neck emphysema, post-operative bleeding, mediastinitis or aspiration pneumonia were observed in the present series. A swallow survey was obtained by telephone: 85% of patients reported improved swallowing (without symptoms in 11 and with moderate dysphagia in 7). Based on the present series, endoscopic CO(2) laser cricopharyngeal myectomy for Zenker's diverticulum can be considered an effective and safe procedure, with reduced hospitalization time and complication rate.

Narrow band imaging and high definition television in evaluation of oral and oropharyngeal squamous cell cancer: a prospective study.

Narrow band imaging (NBI) is an optical technique in which filtered light enhances superficial neoplasms based on their neoangiogenic pattern. The accuracy of NBI can be augmented by combining it with high definition television (HDTV). The aim of this study was to prospectively assess the diagnostic value of NBI in combination with HDTV in evaluation of oral (O) and oropharyngeal (OP) squamous cell carcinoma (SCC). Between April 2007 and December 2009, we analyzed 96 patients who were divided into 2 groups: Group A included 35 patients previously biopsied and diagnosed with OSCC or OPSCC and subjected to pre- and intraoperative HDTV white light (WL) and HDTV NBI endoscopy; Group B included 61 subjects already treated for OSCC or OPSCC and followed-up with HDTV WL and HDTV NBI. Fourteen of 35 (40%) patients in Group A showed adjunctive findings with NBI compared to standard WL. All of these findings were histologically confirmed. Twelve of 61 (20%) patients in Group B showed positive NBI findings, which were all confirmed by histology. The sensitivity, specificity, positive, negative predictive values, and accuracy for HDTV WL were 51%, 100%, 100%, 87%, and 68%, respectively, whilst for HDTV NBI were 96%, 100%, 100%, 93%, and 97%, respectively. Overall, 26 of 96 (27%) patients had a diagnostic advantage in applying NBI and HDTV: 6 patients received a diagnosis of recurrence and 1 of persistence after previous treatments; 5 showed a metachronous tumour; in 4 a synchronous tumour was diagnosed; 9 lesions were upstaged; in 1 patient previously diagnosed with an unknown primary by fine needle aspiration cytology on the neck, an anterior tonsillar pillar cancer was identified.

The photon analysis, delivery, and reduction system at the FERMI@Elettra free electron laser user facility.

The FERMI@Elettra free electron laser (FEL) user facility is currently under construction at the Sincrotrone Trieste laboratory in Trieste (Italy). It will cover the wavelength range from 100 to about 5 nm in the fundamental and 3 or 1 nm using the third harmonic. We report the layout of the photon beam diagnostics section, the radiation transport system to the experimental area, and the photon beam distribution system. Due to the peculiar characteristics of the emitted FEL radiation (high peak power, short pulse length, and statistical variation of the emitted intensity and distribution), the realization of the diagnostics system is particularly challenging. The end users are interested in parameters such as the radiation pulse intensity and spectral distribution, as well as in the possibility to attenuate the intensity. In order to accomplish these tasks, a photon analysis, delivery, and reduction system is now under development and construction and is presented here. This system will work on-line producing pulse-resolved information and will let users keep track of the photon beam parameters during the experiments.

Advanced photoelectric effect experiment beamline at Elettra: A surface science laboratory coupled with Synchrotron Radiation.

We report the main characteristics of the advanced photoelectric effect experiments beamline, operational at Elettra storage ring, featuring a fully independent double branch scheme obtained by the use of chicane undulators and able to keep polarization control in both linear and circular mode. The paper describes the novel technical solutions adopted, namely, (a) the design of a quasiperiodic undulator resulting in optimized suppression of higher harmonics over a large photon energy range (10-100 eV), (b) the thermal stability of optics under high heat load via cryocoolers, and (c) the end station interconnected setup allowing full access to off-beam and on-beam facilities and, at the same time, the integration of users' specialized sample growth chambers or modules.

Narrow-band imaging: a new tool for evaluation of head and neck squamous cell carcinomas. Review of the literature.

Head and neck squamous cell carcinoma of the upper aerodigestive tract is well known for its frequently late presentation and diagnosis at an advanced stage. In addition, it is well recognized that it may arise in multiple sites, either synchronously or metachronously. Thus it should be imperative to endoscopically screen the upper aerodigestive tract of patients at risk for head and neck squamous cell carcinoma with a new diagnostic tool, especially due to the fact that early lesions are very difficult to detect even by multiple passes with a standard endoscopy, if they are < or = 1 cm in diameter. Lugol chromoendoscopy, which is mainly used in the oesophagus, is not suitable for the head and neck region due to severe mucosal irritation. Herein, narrow-band imaging is described, a diagnostic tool already proved as a useful screening method in other endoscopic fields, and its application in the early detection of head and neck squamous cell carcinoma is reviewed, as reported by previous studies in the otolaryngologic literature. Narrow-band imaging relies on the principle of depth of penetration of light, with the narrow-band blue light having a short wavelength (415 nm) penetrating into the mucosa and highlighting the superficial vasculature. Furthermore, the blue filter is designed to correspond to the peak absorption spectrum of haemoglobin to enhance the image of capillary vessels on surface mucosa. Thus, superficial mucosal lesions that would be missed by regular white light endoscopy, are identified, in view of their neoangiogenetic pattern of vasculature, using the blue light of the narrow-band imaging. Narrow-band imaging has been used extensively in the lower aerodigestive system, yet there are only 2 reports of applications in the region of the head and neck, specifically the oropharynx and the hypopharynx. However, these are not the only sites that can benefit from narrow-band imaging. Herewith, the uses and importance are highlighted of narrow-band imaging as a future diagnostic tool in otolaryngology, in the pre-, intra- and post-operative settings.

Carcinoma cuniculatum of the larynx: a rare clinicopathological entity.

OBJECTIVE: Carcinoma cuniculatum is a rare variant of low-grade squamous cell carcinoma. We report the second case of carcinoma cuniculatum of the larynx in the literature. METHOD: Case report and review of the world literature concerning carcinoma cuniculatum of the larynx and upper aerodigestive tract. RESULTS: A histologically proven carcinoma cuniculatum of the larynx is reported in a 72-year-old man. The patient underwent a supracricoid laryngectomy with crico-hyoidopexy, and was free of disease at 70 months after surgery. We emphasise the clinical presentation, histology and therapeutic approach of this rare tumour. CONCLUSION: To our knowledge this is the second report in the world literature of carcinoma cuniculatum of the larynx. Carcinoma cuniculatum of the larynx must be considered as a distinct 'clinicopathological entity' and close cooperation between the clinician and the pathologist is essential for the correct diagnosis of these tumour as regards to the correct classification and therapy.

Properties of Thermus aquaticus beta-NADH oxidase immobilised on various supports.

beta-NADH oxidase purified from Thermus aquaticus was covalently immobilised on various solid supports. The preparations obtained were compared with the soluble enzyme for activity and kinetic properties. Activated glutaryl-PVA was found to be the best support. The immobilised enzyme was less stable at high temperatures than the soluble enzyme. No differences could be detected in the presence of organic solvents.

Effect of NAD(P)H:quinone oxidoreductase on tyrosinase-mediated oxidation of opioid neuropeptides Leu-enkephalin and Met-enkephalin.

In vitro experiments are reported showing that NAD(P)H:(quinone acceptor) oxidoreductase (QR), purified from Glycine max seedlings, reduces Leu- and Met-enkephalin-tyrosinase oxidation products, in the presence of NADH or NADPH. QR was not capable to catalyze the reduction of N-acetyl-dopaquinone formed by the cation of mushroom tyrosinase on N-acetyl-L-tyrosine, while it was able to reduce dopachrome. The results support the hypothesis that QR can inhibit the formation of melanin-like compounds, as catalyzed by the action of tyrosinase on Leu-enkephalin and Met-enkephalin. It is proposed that, in the presence of NAD(P)H as the electron donor, the inhibition occurs by the specific conversion of the dopachrome-derivative into the reduced precursor, leucodopachrome-derivative.

Thermostable reduced nicotinamide adenine dinucleotide oxidase: application to amperometric enzyme assay.

The use in amperometric enzyme assays of a highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), from the thermophilic organism Thermus aquaticus is described. The enzyme catalyses the oxidation of reduced nicotinamide adenine dinucleotide with concomitant two-electron reduction of dioxygen to hydrogen peroxide. In addition the enzyme used a substituted ferrocene as an alternative mediator of electron transfer. Hydrogen peroxide was detected at +650 mV vs Ag/AgCl at a platinum electrode. The current produced by oxidation of hydrogen peroxide was directly proportional to NADH concentration. The enzyme was used in solution to reoxidize enzymatically generated NADH and served as a basis for amperometric enzyme amplification systems for immunoassay as well as for the detection of substrate concentration for oxidoreductase enzymes. In the presence of alcohol dehydrogenase a rapid production of current occurred upon addition of ethanol over a clinically significant range. Thermus aquaticus NADH oxidase appears to be ideally suited for future exploitation in amperometric sensors for oxidoreductase substrates, offering a number of advantages over previously reported methods.

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation.

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.

Preparation of reduced bovine Cu,Zn superoxide dismutase.

N.m.r. and e.p.r. were used to measure the oxidation state of copper in Cu,Zn superoxide dismutase treated with reducing agents such as NaBH4, K4Fe(CN)6, Na2S2O4 and H2O2. The activity and the electrophoretic pattern of the treated enzyme were also studied. On the basis of the reducing ability and of the absence of inactivating effects, NaBH4 was the most suitable reducer of those tested. Some characteristics of the reduction of superoxide dismutase by NaBH4 were further investigated. The results obtained indicate that NaBH4 can be used to prepare, in a few minutes, solutions of completely reduced enzyme without any apparent change of the activity and of the structure.

Co(II) derivatives of Cu,Zn-superoxide dismutase with the cobalt bound in the place of copper. A new spectroscopic tool for the study of the active site.

Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.

Electrostatic interactions in Cu,Zn superoxide dismutase. Effects of Ca(II) and of anions not binding to the copper.

Cu,Zn superoxide dismutase (EC 1.15.1.1) from bovine erythrocyte was found to enhance Tb(III) luminescence in a way suggestive of the presence of specific sites binding calcium. Binding of Ca(II) had no effect on the enzyme activity. However, the low-temperature EPR spectra of the enzyme-bound copper were modified into a more axial line shape by Ca(II) and Tb(III), but not by other cations. This effect was not observed by room-temperature EPR and was interpreted as being due to a long-range conformational effect on the copper-binding site occurring on freezing when specific charged amino acid side-chains are neutralized. This interpretation is supported by similar effects observed with anions that neither have inhibitory effect on the activity nor bind to the copper. These results indicate the presence of specific electrostatic interactions of the protein moiety of Cu,Zn superoxide dismutase and the occurrence of changes of the symmetry of the copper site when the protein is frozen under certain conditions.

Reduced anion binding and anion inhibition in Cu, Zn superoxide dismutase chemically modified at lysines without alteration of the rhombic distortion of the copper site.

The spectroscopic binding constant (visible absorption and EPR spectra) and the catalytic inhibition constant of N-3 and CN- were measured for bovine Cu, Zn superoxide dismutase chemically modified at all lysines by either succinylation or carbamoylation. These modifications partially inactivate the enzyme (10% and 50% residual activity respectively) but leave the native rhombic geometry of the copper site unaffected. It could thus be shown that the observed reduction of anion affinity of the lysines-modified proteins is related to the decreased positive charge of the protein.

Carbamoylation of Cu,Zn-superoxide dismutase by cyanate. Role of lysines in the enzyme action.

Reaction with cyanate leads to a reversible change of the EPR spectrum of Cu,Zn-superoxide dismutase and to time-dependent carbamoylation of the lysine residues of the enzyme, producing a stable covalent derivative with more negative charge. The carbamoylated enzyme is less active than the native enzyme in spite of unaltered EPR spectra. The extent of this inactivation is much less when the enzyme activity is measured at low ionic strength. These results show that integrity of the active site is not the sole factor playing a role in the enzyme mechanism and that the ionic strength effect is related to electrostatic interactions between O-2 and surface charges of the protein.

Evidence for catalytic dismutation of superoxide by cobalt(II) derivatives of bovine superoxide dismutase in aqueous solution as studied by pulse radiolysis.

By using the technique of pulse radiolysis to generate O2-., it is demonstrated that Co(II) derivatives of bovine superoxide dismutase in which the copper alone and both the copper and zinc of the enzyme have been substituted by Co(II), resulting in (Co,Zn)- and (Co,Co)-proteins, are capable of catalytically dismutating O2-. with 'turnover' rate constants of 4.8 X 10(6) dm3.s-1.mol-1 and 3.1 X 10(6) dm3.s-1.mol-1 respectively. The activities of the proteins are independent of the pH (7.4-9.4) and are about three orders of magnitude less than that of the native (Cu,Zn)-protein. The rate constants for the initial interaction of O2-. with the Co-proteins were determined to be (1.5-1.6) X 10(9) dm3.s-1.mol-1; however, in the presence of phosphate, partial inhibition is apparent [k approximately (1.9-2.3) X 10(8) dm3.s-1.mol-1]. To account for the experimental observations, two reaction schemes are presented, involving initially either complex-formation or redox reactions between O2-. and Co(II). This is the first demonstration that substitution of a metal into the vacant copper site of (Cu,Zn)-protein results in proteins that retain superoxide dismutase activity.