New methods of structure refinement for macromolecular structure determination by NMR

Recent advances in multidimensional NMR methodology have permitted solution structures of proteins in excess of 250 residues to be solved. In this paper, we discuss several methods of structure refinement that promise to increase the accuracy of macromolecular structures determined by NMR. These methods include the use of a conformational database potential and direct refinement against three-bond coupling constants, secondary 13C shifts, 1H shifts, T1/T2 ratios, and residual dipolar couplings. The latter two measurements provide long range restraints that are not accessible by other solution NMR parameters.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain ( DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-KB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone N-15 NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha 2-alpha 3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha 2-alpha 3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha 2-alpha 3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Refinement of Rapid PCC Coarse Aggregate Testing Program, HR-1071, 1999

The characterization and categorization of coarse aggregates for use in portland cement concrete (PCC) pavements is a highly refined process at the Iowa Department of Transportation. Over the past 10 to 15 years, much effort has been directed at pursuing direct testing schemes to supplement or replace existing physical testing schemes. Direct testing refers to the process of directly measuring the chemical and mineralogical properties of an aggregate and then attempting to correlate those measured properties to historical performance information (i.e., field service record). This is in contrast to indirect measurement techniques, which generally attempt to extrapolate the performance of laboratory test specimens to expected field performance. The purpose of this research project was to investigate and refine the use of direct testing methods, such as X-ray analysis techniques and thermal analysis techniques, to categorize carbonate aggregates for use in portland cement concrete. The results of this study indicated that the general testing methods that are currently used to obtain data for estimating service life tend to be very reliable and have good to excellent repeatability. Several changes in the current techniques were recommended to enhance the long-term reliability of the carbonate database. These changes can be summarized as follows: (a) Limits that are more stringent need to be set on the maximum particle size in the samples subjected to testing. This should help to improve the reliability of all three of the test methods studied during this project. (b) X-ray diffraction testing needs to be refined to incorporate the use of an internal standard. This will help to minimize the influence of sample positioning errors and it will also allow for the calculation of the concentration of the various minerals present in the samples. (c) Thermal analysis data needs to be corrected for moisture content and clay content prior to calculating the carbonate content of the sample.

Fecal water as a non-invasive biomarker in nutritional intervention: comparison of preparation methods and refinement of different endpoints

The assessment of cellular effects by the aqueous phase of human feces (fecal water, FW) is a useful biomarker approach to study cancer risks and protective activities of food. In order to refine and develop the biomarker, different protocols of preparing FW were compared. Fecal waters were prepared by 3 methods: (A) direct centrifugation; (B) extraction of feces in PBS before centrifugation; and (C) centrifugation of lyophilized and reconstituted feces. Genotoxicity was determined in colon cells using the Comet assay. Selected samples were investigated for additional parameters related to carcinogenesis. Two of 7 FWs obtained by methods A and B were similarly genotoxic. Method B, however, yielded higher volumes of FW, allowing sterile filtration for long-term culture experiments. Four of 7 samples were non-genotoxic when prepared according to all 3 methods. FW from lyophilized feces and from fresh samples were equally genotoxic. FWs modulated cytotoxicity, paracellular permeability, and invasion, independent of their genotoxicity. All 3 methods of FW preparation can be used to assess genotoxicity. The higher volumes of FWobtained by preparation method B greatly enhance the perspectives of measuring different types of biological parameters and using these to disclose activities related to cancer development.

Envy: A Psychometric Refinement of the Construct

The noticeable differences in the theoretical and operative definitions of envy across studies and approaches have produced a fragmentary representation and understanding of the envious emotion. The present dissertation aimed to clarify the inherent nature of the construct of envy through the integration of findings from three independent studies. We focused on malicious envy, and investigated it from both a dispositional and an episodic perspective. Studies 1 and 2 investigated the dimensionality of envy as a stable individual characteristic and an episodic emotional state, respectively. In order to elicit episodic envy, a scenario-based experiment was conducted. Results indicated that, in both its dispositional and episodic facets, envy is a bidimensional construct composed by an inner-directed dimension of inferiority and helplessness, and an outer-directed dimension of feelings of anger and ill will. Moreover, findings from Study 1 allowed to establish boundaries between envy and competing constructs that have often been included in conceptualizations of envy. The psychometrically validated definition of envy provided by Studies 1 and 2 represents a valuable contribution to empirical research. Implications for envy research concern the promotion of a shared operationalization of envy in future studies, which will arguably facilitate the comparison of findings between studies and between approaches. Study 3 examined the mechanisms through which dispositional envy affects individuals’ social adjustment and psychological wellbeing. Findings revealed that the detrimental effects of envy on perceived social support and subjective well-being are mostly mediated by other personal characteristics, such as neuroticism and self-esteem. By reducing global self-esteem, the envious disposition may damage supportive social networks via antisocial direct and indirect behaviors that may arise from envy and that are likely to drive others away. On the other hand, by damaging both emotional stability and self-worth, dispositional envy leads to reduced subjective well-being. Implications for clinical practice are discussed.

Global non-dynamic refinement of radial orbit error for altimetric earth satellites

The accuracy of altimetrically derived oceanographic and geophysical information is limited by the precision of the radial component of the satellite ephemeris. A non-dynamic technique is proposed as a method of reducing the global radial orbit error of altimetric satellites. This involves the recovery of each coefficient of an analytically derived radial error correction through a refinement of crossover difference residuals. The crossover data is supplemented by absolute height measurements to permit the retrieval of otherwise unobservable geographically correlated and linearly combined parameters. The feasibility of the radial reduction procedure is established upon application to the three day repeat orbit of SEASAT. The concept of arc aggregates is devised as a means of extending the method to incorporate longer durations, such as the 35 day repeat period of ERS-1. A continuous orbit is effectively created by including the radial misclosure between consecutive long arcs as an infallible observation. The arc aggregate procedure is validated using a combination of three successive SEASAT ephemerides. A complete simulation of the 501 revolution per 35 day repeat orbit of ERS-1 is derived and the recovery of the global radial orbit error over the full repeat period is successfully accomplished. The radial reduction is dependent upon the geographical locations of the supplementary direct height data. Investigations into the respective influences of various sites proposed for the tracking of ERS-1 by ground-based transponders are carried out. The potential effectiveness on the radial orbital accuracy of locating future tracking sites in regions of high latitudinal magnitude is demonstrated.

Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Direct Adhesive Pin-retained Restorations For Severely Worn Dentition Treatment: A 1.5-year Follow-up Report.

Excessive occlusal surface wear can result in occlusal disharmony, functional and esthetic impairment. As a therapeutic approach, conventional single crowns have been proposed, but this kind of treatment is complex, highly invasive and expensive. This case report describes the clinical outcomes of an alternative minimally invasive treatment based on direct adhesive-pin retained restorations. A 64-year-old woman with severely worn dentition, eating problems related to missing teeth and generalized tooth hypersensitivity was referred for treatment. Proper treatment planning based on the diagnostic wax-up simulation was used to guide the reconstruction of maxillary anterior teeth with direct composite resin over self-threading dentin pins. As the mandibular remaining teeth were extremely worn, a tooth-supported overdenture was installed. A stabilization splint was also used to protect the restorations. This treatment was a less expensive alternative to full-mouth rehabilitation with positive esthetic and functional outcomes after 1.5 years of follow-up.

Direct And Non-destructive Proof Of Authenticity For The 2nd Generation Of Brazilian Real Banknotes Via Easy Ambient Sonic Spray Ionization Mass Spectrometry.

Using a desorption/ionization technique, easy ambient sonic-spray ionization coupled to mass spectrometry (EASI-MS), documents related to the 2nd generation of Brazilian Real currency (R$) were screened in the positive ion mode for authenticity based on chemical profiles obtained directly from the banknote surface. Characteristic profiles were observed for authentic, seized suspect counterfeit and counterfeited homemade banknotes from inkjet and laserjet printers. The chemicals in the authentic banknotes' surface were detected via a few minor sets of ions, namely from the plasticizers bis(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP), most likely related to the official offset printing process, and other common quaternary ammonium cations, presenting a similar chemical profile to 1st-generation R$. The seized suspect counterfeit banknotes, however, displayed abundant diagnostic ions in the m/z 400-800 range due to the presence of oligomers. High-accuracy FT-ICR MS analysis enabled molecular formula assignment for each ion. The ions were separated by 44 m/z, which enabled their characterization as Surfynol® 4XX (S4XX, XX=40, 65, and 85), wherein increasing XX values indicate increasing amounts of ethoxylation on a backbone of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (Surfynol® 104). Sodiated triethylene glycol monobutyl ether (TBG) of m/z 229 (C10H22O4Na) was also identified in the seized counterfeit banknotes via EASI(+) FT-ICR MS. Surfynol® and TBG are constituents of inks used for inkjet printing.

Fast And Direct Na And K Determination In Table, Marine, And Low-sodium Salts By X-ray Fluorescence And Chemometrics.

X-ray fluorescence (XRF) is a fast, low-cost, nondestructive, and truly multielement analytical technique. The objectives of this study are to quantify the amount of Na(+) and K(+) in samples of table salt (refined, marine, and light) and to compare three different methodologies of quantification using XRF. A fundamental parameter method revealed difficulties in quantifying accurately lighter elements (Z < 22). A univariate methodology based on peak area calibration is an attractive alternative, even though additional steps of data manipulation might consume some time. Quantifications were performed with good correlations for both Na (r = 0.974) and K (r = 0.992). A partial least-squares (PLS) regression method with five latent variables was very fast. Na(+) quantifications provided calibration errors lower than 16% and a correlation of 0.995. Of great concern was the observation of high Na(+) levels in low-sodium salts. The presented application may be performed in a fast and multielement fashion, in accordance with Green Chemistry specifications.

Effect of staining solutions and repolishing on color stability of direct composites

OBJECTIVES: The purpose of this study was to assess the color change of three types of composite resins exposed to coffee and cola drink, and the effect of repolishing on the color stability of these composites after staining. MATERIALS AND METHODS: Fifteen specimens (15 mm diameter and 2 mm thick) were fabricated from microhybrid (Esthet-X; Dentsply and Filtek Z-250; 3M ESPE) and high-density hybrid (Surefil; Dentsply) composites, and were finished and polished with aluminum oxide discs (Sof-Lex; 3M ESPE). Color of the specimens was measured according to the CIE L*a*b* system in a refection spectrophotometer (PCB 6807; BYK Gardner). After baseline color measurements, 5 specimens of each resin were immersed in different staining solutions for 15 days: G1 - distilled water (control), G2 - coffee, G3 - cola soft drink. Afterwards, new color measurement was performed and the specimens were repolished and submitted to new color reading. Color stability was determined by the difference (ΔE) between the coordinates L*, a*, and b* obtained from the specimens before and after immersion into the solutions and after repolishing. RESULTS: There was no statistically signifcant difference (ANOVA, Tukey's test; p>0.05) among the ΔE values for the different types of composites after staining or repolishing. For all composite resins, coffee promoted more color change (ΔE>3.3) than distilled water and the cola soft drink. After repolishing, the ΔE values of the specimens immersed in coffee decreased to clinically acceptable values (ΔE<3.3), but remained signifcantly higher than those of the other groups. CONCLUSIONS: No signifcant difference was found among composite resins or between color values before and after repolishing of specimens immersed in distilled water and cola. Immersing specimens in coffee caused greater color change in all types of composite resins tested in this study and repolishing contributed to decrease staining to clinically acceptable ΔE values.

Heat treatment of a direct composite resin: influence on flexural strength

The purpose of this study was to evaluate the flexural strength of a direct composite, for indirect application, that received heat treatment, with or without investment. One indirect composite was used for comparison. For determination of the heat treatment temperature, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed, considering the initial weight loss temperature and glass transition temperature (Tg). Then, after photoactivation (600 mW/cm² - 40 s), the specimens (10 x 2 x 2 mm) were heat-treated following these conditions: 170ºC for 5, 10 or 15 min, embedded or not embedded in investment. Flexural strength was assessed as a means to evaluate the influence of different heat treatment periods and investment embedding on mechanical properties. The data were analyzed by ANOVA and Tukey's test (α = 0.05). TGA showed an initial weight loss temperature of 180ºC and DSC showed a Tg value of 157°C. Heat treatment was conducted in an oven (Flli Manfredi, Italy), after 37°C storage for 48 h. Flexural strength was evaluated after 120 h at 37°C storage. The results showed that different periods and investment embedding presented similar statistical values. Nevertheless, the direct composite resin with treatments presented higher values (178.7 MPa) compared to the indirect composite resin (146.0 MPa) and the same direct composite submitted to photoactivation only (151.7 MPa). Within the limitations of this study, it could be concluded that the heat treatment increased the flexural strength of the direct composite studied, leading to higher mechanical strength compared to the indirect composite.

Flexural strength and hardness of direct and indirect composites

The objective of this study was to evaluate the flexural strength (σf) and hardness (H) of direct and indirect composites, testing the hypotheses that direct resin composites produce higher σf and H values than indirect composites and that these properties are positively related. Ten bar-shaped specimens (25 mm x 2 mm x 2 mm) were fabricated for each direct [D250 - Filtek Z250 (3M-Espe) and D350 - Filtek Z350 (3M-Espe)] and indirect [ISin - Sinfony (3M-Espe) and IVM - VitaVM LC (Vita Zahnfabrik)] materials, according to the manufacturer's instructions and ISO4049 specifications. The σf was tested in three-point bending using a universal testing machine (EMIC DL 2000) at a crosshead speed of 0.5 mm/min (ISO4049). Knoop hardness (H) was measured on the specimens' fragments resultant from the σf test and calculated as H = 14.2P/l², where P is the applied load (0.1 kg; dwell time = 15 s) and l is the longest diagonal of the diamond shaped indent (ASTM E384). The data were statistically analyzed using Anova and Tukey tests (α = 0.05). The mean σf and standard deviation values (MPa) and statistical grouping were: D250 - 135.4 ± 17.6a; D350 - 123.7 ± 11.1b; ISin - 98.4 ± 6.4c; IVM - 73.1 ± 4.9d. The mean H and standard deviation values (kg/mm²) and statistical grouping were: D250 - 98.12 ± 1.8a; D350 - 86.5 ± 1.9b; ISin - 28.3 ± 0.9c; IVM - 30.8 ± 1.0c. The direct composite systems examined produce higher mean σf and H values than the indirect composites, and the mean values of these properties were positively correlated (r = 0.91), confirming the study hypotheses.

Rat pinealectomy: a modified direct visual approach

PURPOSE: To report a new, direct visual approach for rat pinealectomy. METHODS: Eighty adult female rats (Rattus norvegicus albinus EPM-1 strain) were weighted and anesthetized intraperitoneally with 15 mg/kg xylazine and 30 mg/kg ketamine. The animal was fastened to a dissection table, an incision was made in the skin and the subcutaneous tissue, bringing the lambda into view. The skullcap was opened with a dental drill, bringing the cerebral hemispheres and the superior sagittal sinus into view. The pineal gland, located under the venous sinus, was removed in a single piece using tweezers. Next, the bone fragment was returned to its place and the surgical layers were sutured. RESULTS: This new technique is easy to be done, avoids bleedings and removes only the pineal gland without damage to the remaining encephalon. In addition it makes possible the achievement of a sham surgery, allowing the pineal gland to remain intact. CONCLUSION: The proposed technique intends to facilitate studies aiming to better understanding the complexity and importance of the pineal gland on reproductive and other body systems.