New insights on ocular surface disease in patients with atopic dermatitis treated with dupilumab.

Our study sought to describe ocular surface alterations at baseline and after 4 months of dupilumab treatment in patients with severe AD. Our findings highlight that all 25 patients showed ocular surface alterations prior to dupilumab treatment. Dupilumab may cause the worsening of clinical or subclinical pre-existing ocular alterations belonging to the spectrum of AKC.

The efficiency of quartz addition on electric arc furnace (EAF) carbon steel slag stability.

Electric arc furnace slag (EAF) has the potential to be re-utilized as an alternative to stone material, however, only if it remains chemically stable on contact with water. The presence of hydraulic phases such as larnite (2CaO SiO2) could cause dangerous elements to be released into the environment, i.e. Ba, V, Cr. Chemical treatment appears to be the only way to guarantee a completely stable structure, especially for long-term applications. This study presents the efficiency of silica addition during the deslagging period. Microstructural characterization of modified slag was performed by SEM and XRD analysis. Elution tests were performed according to the EN 12457-2 standard, with the addition of silica and without, and the obtained results were compared. These results demonstrate the efficiency of the inertization process: the added silica induces the formation of gehlenite, which, even in caustic environments, does not exhibit hydraulic behaviour.

Validation and attempts of revision of the MDS-recommended tests for the screening of Parkinson's disease dementia.

The Movement Disorders Society (MDS) formulated diagnostic criteria and assessment guidelines for the screening of dementia in Parkinson's disease (PD). We carried out a validation of the cognitive measures suggested in the screening algorithm (i.e. the Mini Mental State Examination - MMSE - total score, serial 7s subtraction, 3-word recall, pentagons copy, and one minute letter fluency) in 86 patients with PD. Thirty-six percent of participants were diagnosed with dementia using the MDS algorithm, but with the Dementia Rating Scale instead of the MMSE. The original MDS procedure misclassified 11 patients (12.8%) as false negatives and 3 (3.5%) as false positives, leading to 65% sensitivity and 95% specificity. The main reason for misdiagnoses was insensitivity of the MMSE total score. Three attempts were made to reach a better screening performance, which warrants high sensitivity more than high specificity: 1. exclusion of the MMSE total score as a diagnostic requirement; 2. determination of a better cut off through Receiver Operating Characteristic curve analysis; 3. replacement of the MMSE with the equally undemanding, but more PD-specific, Mini Mental Parkinson. The first two strategies generally yielded high sensitivity, but poor specificity. The best outcome was achieved using a Mini Mental Parkinson total score <27 as cognitive criterion: sensitivity was 87% and negative predictive value was 90%; however, specificity was only 67%. Our findings seem to suggest that MDS practical guidelines are specific, but might benefit from the use of more PD-oriented tools than the MMSE in terms of sensitivity.

Psychometric properties of the Italian version of the Scales for Outcomes in Parkinson's disease- Cognition (SCOPA-Cog).

The Scales for Outcomes in Parkinson's disease-Cognition (SCOPA-Cog) has been shown to be a clinimetrically rigorous and valid instrument for a disease-oriented neuropsychological assessment of Parkinson's disease (PD) patients. In the present study we evaluated the psychometric properties of the Italian version of the SCOPA-Cog in 121 PD patients. The scale explores memory, attention, and executive and visuospatial functions and takes approximately 20 minutes to administer. Data distribution (skewness= -0.23) and internal consistency (Cronbach's alpha= 0.78) were satisfactory. Standard error of measurement was 3.42. The outcome was significantly worse in patients with an abnormal Psychometric properties of the Italian version of the Scales for Outcomes in Parkinson's disease-Cognition (SCOPA-Cog) score on the Dementia Rating Scale (DRS) (SCOPACog mean score 14.6 ± 5.1 out of a total of 43) with respect to cognitively intact subjects (24.2 ± 4.3) (p<0.0001). The DRS showed good convergent validity (Spearman rho= 0.77, p<0.0001), and a high coefficient of variation (= 0.34). These findings support the goodness of the Italian SCOPA-Cog in terms of metrics and validity.

Diagnosis of possible mild cognitive impairment in Parkinson's disease: validity of the SCOPA-Cog.

The detection of cognitive decline in Parkinson's disease (PD), at the Mild Cognitive Impairment (MCI) stage, has prognostic and treatment implications. The Movement Disorders Society (MDS) has recently published criteria and guidelines for the diagnosis of possible and probable PD-MCI. In the present study we assessed the ability of the Scales for Outcomes in Parkinson's disease-Cognition (SCOPA-Cog) to discriminate possible PD-MCI cases from patients with PD-dementia (PDD) and from cognitively intact PD subjects. Hundred-and-thirteen consecutive PD patients underwent the MMSE, the Dementia Rating Scale and an interview on independence in daily living, and were classified as cognitively intact (n = 49), or as possible PD-MCI (n = 33) or PDD (n = 31), according to MDS criteria. Logistic regression analysis was carried out with PD-MCI diagnosis (yes/no) as an outcome variable, and age, education and the SCOPA-Cog total score as covariates. Classification of cases according to the regression model was used for constructing Receiver Operating Characteristic (ROC) curves. Area Under the Curve (AUC) was 0.92 [95% CI 0.86-0.98], for the differential diagnosis between PD-MCI and cognitively normal patients, and 0.97 [95% CI 0.80-1.00], for the differential diagnosis between PD-MCI and PDD. Sensitivity and specificity were 90% and 73% for the PD-MCI versus no cognitive impairment differentiation, at the cutpoint ≥24, and 93% and 97% for the PD-MCI versus PDD discrimination, at the cutpoint ≥17. The SCOPA-Cog is a quick and psychometrically sound PD-specific scale. Our findings support its use for the screening of possible PD-MCI.

Validity and metric of MiniMental Parkinson and MiniMental State Examination in Parkinson's disease.

The MiniMental Parkinson (MMP) has been derived from the MiniMental State Examination (MMSE) for the screening of cognitive impairment in Parkinson's disease by adding subtests that were focused on executive and visuo-spatial impairment more than on memory or language deficits. In this multicenter study, the psychometric and validity properties of the MMP have been evaluated in 69 cognitively intact and 52 cognitively impaired patients with Parkinson's disease, classified according to their performance at the Dementia Rating Scale. The MMP showed better metrics and convergent validity, and higher screening ability. However, its performance was not fully satisfying in terms of data distribution, coefficient of variation and specificity, and Receiver Operating Characteristic curves did not show clear cut superiority of either scale at their best sensitivity-specificity trade off. The MMP seems to be slightly preferable to the MMSE only at a cut off that favours sensitivity with respect to specificity, for screening purposes. The test is simple and quick, but has limitations in terms of validity.

Sequential combined treatment with intravitreal bevacizumab and photodynamic therapy for retinal angiomatous proliferation.

PURPOSE: To study visual and anatomical outcomes of sequenced combined therapy using intravitreal bevacizumab followed by photodynamic therapy (PDT) in eyes with retinal angiomatous proliferation (RAP). Safety and rate of intravitreal injections were also evaluated. METHODS: We conducted a prospective non-comparative pilot study of consecutive patients newly diagnosed with RAP. PDT guided by indocyanine green (ICG) angiography was applied 8+/-2 days after the intravitreal bevacizumab (1.25 mg) injection. At baseline and every month after the injection, best-corrected visual acuity (BCVA) measurement, complete eye examination including dynamic fluorescein and ICG angiography, and optical coherence tomography (OCT) were performed. RESULTS: In all, 21 eyes of 18 patients with RAP were enrolled. The mean age was 77 (range 65-86) years. Mean visual acuity at baseline was 0.63+/-0.25 logMAR. After treatment BCVA showed no statistically significant differences between each visit (P=0.10, ANOVA). At 9 months, the BCVA improved by three or more lines in three eyes (14%), remained stable in twelve eyes (57%), and worsened in six eyes (29%). Foveal thickness decreased significantly between baseline and all the follow-up visits (P<0.01, ANOVA). A total of 36 intravitreal injections were given during the study with a mean of 1.7 injections per eye (range 1-3 injections per eye). No ocular or systemic adverse events were reported. CONCLUSION: A possible synergistic effect may arise from the combination of intravitreal bevacizumab with PDT for the treatment of RAP. These findings also suggest a possible benefit of combo therapy in the rate of intravitreal re-injections.

Solution structure of the Grb2 N-terminal SH3 domain complexed with a ten-residue peptide derived from SOS: direct refinement against NOEs, J-couplings and 1H and 13C chemical shifts.

Refined ensembles of solution structures have been calculated for the N-terminal SH3 domain of Grb2 (N-SH3) complexed with the ac-VPPPVPPRRR-nh2 peptide derived from residues 1135 to 1144 of the mouse SOS-1 sequence. NMR spectra obtained from different combinations of both 13C-15N-labeled and unlabeled N-SH3 and SOS peptide fragment were used to obtain stereo-assignments for pro-chiral groups of the peptide, angle restraints via heteronuclear coupling constants, and complete 1H, 13C, and 15N resonance assignments for both molecules. One ensemble of structures was calculated using conventional methods while a second ensemble was generated by including additional direct refinements against both 1H and 13C(alpha)/13C(beta) chemical shifts. In both ensembles, the protein:peptide interface is highly resolved, reflecting the inclusion of 110 inter-molecular nuclear Overhauser enhancement (NOE) distance restraints. The first and second peptide-binding sub-sites of N-SH3 interact with structurally well-defined portions of the peptide. These interactions include hydrogen bonds and extensive hydrophobic contacts. In the third highly acidic sub-site, the conformation of the peptide Arg8 side-chain is partially ordered by a set of NOE restraints to the Trp36 ring protons. Overall, several lines of evidence point to dynamical averaging of peptide and N-SH3 side-chain conformations in the third subsite. These conformations are characterized by transient charge stabilized hydrogen bond interactions between the peptide arginine side-chain hydrogen bond donors and either single, or possibly multiple, acceptor(s) in the third peptide-binding sub-site.

In vitro proteolytic activity and active-site identification of the human cytomegalovirus protease.

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in kcat/Km for a maturational peptide (mP4-P8') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of [14C]iodoacetic acid, but when approximately 4.5 mol 14C were incorporated/mol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 3H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.

Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.

NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro activity of the herpes simplex virus type 1 protease with peptide substrates.

Herpes simplex virus type-1 (HSV-1) protease is responsible for proteolytic processing of itself and the virus assembly protein ICP35 (infected cell protein 35). Two proteolytic processing sites within the protease have recently been identified between Ala247 and Ser248 and between Ala610 and Ser611. In this report we demonstrate that peptides corresponding to each of these cleavage sites are substrates for recombinant HSV protease-glutathione S-transferase fusion protein in vitro by high performance liquid chromatography analysis of cleavage reactions. Analysis of the products by fast atom bombardment-mass spectrometry confirmed that cleavage occurred at the expected position between the Ala and Ser residues of the substrate. Peptide cleavage was linear with respect to time and enzyme concentration and proceeded optimally at pH 8.0. A peptide that spans Ala99/Ser100 of the protease but does not correspond to a naturally occurring cleavage site was not a substrate for the protease in vitro confirming that sequence elements outside the conserved dipeptide sequence are required for substrate recognition and cleavage. Identification of P5-P8' as the minimal substrate peptide for the Ala610/Ser611 cleavage site revealed a requirement for residues flanking the conserved core P4-LVNA/S-P1' in substrate recognition and hydrolysis. Kinetic analysis with peptide P5-P8' yielded a Km of 190 microM and kcat of 0.2 min-1. Experiments with a panel of class-specific protease inhibitors were consistent with the protease being a member of the general class of serine proteases.

Micelle-bound conformational preferences of a peptide derived from a murine major histocompatibility complex class I molecule.

Models of the micelle-bound conformation of a 17-residue major histocompatibility complex-derived peptide, [Ala85]Dk(69-85), have been determined by NMR spectroscopy and simulated annealing calculations. This peptide is a truncated, substituted version of Dk(61-85), which is a fragment of the murine major histocompatibility complex class I molecule H-2Dk. Dk(61-85) has been shown to adopt an ordered conformation required for augmentation of insulin-stimulated glucose uptake (Stagsted, J., Baase, W. A., Goldstein, A., and Olsson, L. (1991) J. Biol. Chem. 266, 12844-12847). [Ala85]Dk(69-85) retains full biological activity. Thirty-eight converged NMR structures of [Ala85]Dk(69-85) bound to dodecyl phosphocholine micelles have been generated. The NMR-derived models display a propensity for a type-I beta-bend involving residues 73-76 and an amphipathic helical region involving residues 77-84. CD spectra yield a helical content (8% at 20-25 degrees C) consistent with transient, partial helix formation. The relative orientation of the beta-bend region with respect to the helical region is not well defined by the NMR data. This may reflect true heterogeneity of the micelle-bound conformation. The NMR structures were compared with a model of [Ala85]Dk(69-85) derived from the x-ray coordinates of the human major histocompatibility complex class I allele HLA-Aw68 (Garrett, T. P. J., Saper, M. A., Bjorkmann, P. J., Strominger, T. L., and Wiley, D. C. (1989) Nature 342, 692-696). Structural features that are important for the bioactivity of [Ala85]Dk(69-85) are discussed with reference to reported structure-activity relationships (Stagsted, J., Mapelli, C., Myers, C., Matthews, B. W., Anfinsen, C. B., Goldstein, A., and Olsson, L. (1993) Proc. Natl. Acad. Sci. U.S.A., in press). A general description of the structural properties of the putative receptor site(s) that are likely to be required for binding [Ala85]Dk(69-85) is given.

Amino acid residues essential for biological activity of a peptide derived from a major histocompatibility complex class I antigen.

The stimulatory activity of peptides from the alpha 1 domain of the major histocompatibility complex (MHC) class I antigen on adipose cell glucose transport was previously shown to require a preformed, ordered conformation of the peptide. The two peptides studied previously were Dk-(61-85) (ERETQIAKGNEQSFRVDLRTLLRYY) and Dk-(69-85). We now show that systematic alanine substitution in Dk-(69-85) identifies residues that are essential for biological activity. Ordered structure of the peptides, estimated by circular dichroism, was found in all peptides with activity, but with a complex variety of spectra. Inactive peptides were in either a random coil or an ordered structure. Ordered structure, therefore, is not sufficient for activity. The peptides self-interact in the absence of cells and form aggregates that precipitate upon centrifugation. The tendency to aggregate is correlated with biological potency. Only MHC class I molecules have significant homology to the peptides studied here. The peptide self-interaction suggests that the biological effects in cells, which result from inhibition of receptor and transporter internalization, may be due to the binding (tantamount to self-interaction) of the peptide to the homologous sequences in the alpha 1 domain of the MHC class I molecule.

Synthesis and conformational studies by 1H- and 13C-NMR spectroscopy of a novel, sterically constrained analogue of thyrotropin-releasing hormone.

A novel thyrotropin-releasing hormone (TRH) analogue, [2,4-MePro3]-TRH (2,4-MePro: 2-carboxy-2,4-methanopyrrolidine), has been synthesized using a rapid solid phase peptide synthesis method, and its conformational properties investigated by one- and two-dimensional (2D) nmr spectroscopy and by proton Overhauser measurements. Following a published approach, calibrated interproton Overhauser effects were used together with distance geometry analysis to deduce that the single conformation of the His-2,4-MePro tertiary amide bond is trans in aqueous solution. This conclusion was corroborated by 2D dipolar-correlated (NOESY) spectroscopy. A preferentially extended conformation is indicated by the nmr data, similar to that of TRH. The phi, psi conformational space of 2,4-MePro is, however, significantly different from that of trans proline and the structural consequences of these differences at the C-terminus are discussed. The distribution of histidine side-chain conformations in the TRH analogue was deduced from coupling constants and from the short-range interaction between the imidazole ring and one of the prochiral faces of the 2,4-MePro side chain.

Conformational properties of 2,3-methanopyroglutamic acid in peptides: NMR and x-ray diffraction studies.

The effects of replacing L-pyroglutamic acid with the cyclopropane analogue 2,3-methanopyroglutamic acid (2,3-MeGlp) on conformation and enzymatic stability have been investigated in 2,3-MeGlp-NHMe and the novel thyrotropin releasing hormone (TRH) analogue [2,3-MeGlp1]-TRH by x-ray diffraction and nmr. While 2,3-MeGlp-NHMe adopts a folded conformation (small psi angle) in the solid state, several conformations are available to the molecule in solution. 1H-nmr of the diastereomeric mixture [(+/- )-2,3-MeGlp1]-TRH indicates a close orientation of the pyrrolidone and imidazole rings. The 2,3-MeGlp-His amide bond is considerably more stable to pyroglutamate aminopeptidase than the Glp-His bond in TRH.

Cyclopropane amino acid ester dipeptide sweeteners.

A series of esters of L-aspartyl-1-aminocyclopropane carboxylic acid has been prepared and their sweet tastes determined. The sweetest ester prepared was about 300 times sweeter than sucrose. An attempt to use basic conditions during preparation of the dipeptide allyl ester led to succinimide formation of the aspartyl peptide even though the beta-carboxyl group was protected by a t-butyl ester function. The X-ray structure of the propyl ester (1c) was determined and its conformation is discussed.

Synthesis of four diastereomeric enkephalins incorporating cyclopropyl phenylalanine.

Four diastereomeric D-Ala2, Leu5-enkephalins have been synthesized and their CD spectra and rat brain binding affinities determined. Only the peptides containing Z-cyclopropyl phenylalanine residues showed strong binding affinity. No correlation between CD spectra and bioactivity could be made.