Determination of Biomolecular Interdomain Motions using Nuclear Magnetic Resonance

Biological macromolecules can rearrange interdomain orientations when binding to various partners. Interdomain dynamics serve as a molecular mechanism to guide the transitions between orientations. However, our understanding of interdomain dynamics is limited because a useful description of interdomain motions requires an estimate of the probabilities of interdomain conformations, increasing complexity of the problem.

Staphylococcal protein A (SpA) has five tandem protein-binding domains and four interdomain linkers. The domains enable Staphylococcus aureus to evade the host immune system by binding to multiple host proteins including antibodies. Here, I present a study of the interdomain motions of two adjacent domains in SpA. NMR spin relaxation experiments identified a 6-residue flexible interdomain linker and interdomain motions. To quantify the anisotropy of the distribution of interdomain orientations, we measured residual dipolar couplings (RDCs) from the two domains with multiple alignments. The N-terminal domain was directly aligned by a lanthanide ion and not influenced by interdomain motions, so it acted as a reference frame to achieve motional decoupling. We also applied {\it de novo} methods to extract spatial dynamic information from RDCs and represent interdomain motions as a continuous distribution on the 3D rotational space. Significant anisotropy was observed in the distribution, indicating the motion populates some interdomain orientations more than others. Statistical thermodynamic analysis of the observed orientational distribution suggests that it is among the energetically most favorable orientational distributions for binding to antibodies. Thus, the affinity is enhanced by a pre-posed distribution of interdomain orientations while maintaining the flexibility required for function.

The protocol described above can be applied to other biological systems in general. Protein molecule calmodulin and RNA molecule trans-activation response element (TAR) also have intensive interdomain motions with relative small intradomain dynamics. Their interdomain motions were studied using our method based on published RDC data. Our results were consistent with literature results in general. The differences could be due to previous studies' use of physical models, which contain assumptions about potential energy and thus introduced non-experimental information into the interpretations.

(Table 1) Age determination of sediment core BC5, Golf of Lion

Hitherto unknown abundance peaks of left coiling (l.c.) Neogloboquadrina pachyderma from a Gulf of Lions piston core indicate that abrupt cold spells associated with Atlantic Heinrich events affected the Mediterranean. N. pachyderma (l.c.) is typical of (sub) polar waters in the open ocean. The southern edge of its glacial North Atlantic bioprovince reached south Portugal. Only trace abundances of N. pachyderma (l.c.) are known from Quaternary Mediterranean sediments, suggesting that no significant "invasions" occured via the Strait of Gibraltar. The Gulf of Lions abundance peaks therefore seem to reflect area-specific thriving of a normally rare but indigenous taxon in the western Mediterranean through local favorable habitat development. The general planktonic foraminiferal record suggests that the basic hydrographic regime in the Gulf of Lions, with wintertime deep convective overturn, was relatively stable over the past 60 kyr. Under these conditions, high abundances of N. pachyderma (l.c.) would essentially imply temperature reductions of the order of 5°-8° relative to the present.

Age determination of sediment core GIK16523-1

High-resolution studies of a planktonic foraminifer core record from the South China Sea (SCS) (31KL: 18°45.4'N, 115°52.4'E, water depth 3360 m) reveal changes driven by ice-volume forcings in the climate of the East Asian monsoon in the western Pacific marginal sea during the late Quaternary. The analyses of planktonic foraminifer faunal abundance data from the core indicate significant variations in the relative abundances of the dominant taxa over the past 100,000 years since the isotope stage 5. The transfer function estimates of faunal sea surface temperatures (SST) correlate well with a long-term (104-105 years) trend of global glaciation. About 65,000 years ago, there was an observable change in the mode of SST variability as many low-latitude records have shown. These findings suggest that the SCS surface circulation and the East Asian winter monsoon systems are mainly driven by variations in global glaciation levels. The association of surface ocean cooling in the SCS with global climatic events suggests that fluctuations in the strength of the East Asian winter monsoon may be linked to shifts in the latitudinal position of the westerly winds and the Siberian high-pressure system.

(Figure 4) Stable carbon isotopic values and relative contents of biphytanes and sugars from sediment sample from the Peru margin and from the Pakistan margin

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the d13C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of d13C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the d13C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in 13C relative to the corresponding major biphytane. This 13C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (~15 per mil) than in those from benthic archaea (<7 per mil). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms.

(Table 1) Age determination of sediment cores F2-92-P3 and F2-92-P40

Although climate records from several locations around the world show nearly synchronous and abrupt changes, the nature of the inferred teleconnection is still poorly understood. On the basis of preserved laminations and molybdenum enrichments in open margin sediments we demonstrate that the oxygen content of northeast Pacific waters at 800 m depth during the Bölling-Alleröd warm period (15-13 kyr) was greatly reduced. Existing oxygen isotopic records of benthic and planktonic foraminifera suggest that this was probably due to suppressed ventilation at higher latitudes of the North Pacific. Comparison with ventilation records for the North Atlantic indicates an antiphased pattern of convection relative to the North Pacific over the past 22 kyr, perhaps due to variations in water vapor transport across Central America.

Pose estimation of a humanoid robot using images from a mobile external camera

NOGUEIRA, Marcelo B. ; MEDEIROS, Adelardo A. D. ; ALSINA, Pablo J. Pose Estimation of a Humanoid Robot Using Images from an Mobile Extern Camera. In: IFAC WORKSHOP ON MULTIVEHICLE SYSTEMS, 2006, Salvador, BA. Anais... Salvador: MVS 2006, 2006.

Feature-based camera pose estimation

The estimating of the relative orientation and position of a camera is one of the integral topics in the field of computer vision. The accuracy of a certain Finnish technology company’s traffic sign inventory and localization process can be improved by utilizing the aforementioned concept. The company’s localization process uses video data produced by a vehicle installed camera. The accuracy of estimated traffic sign locations depends on the relative orientation between the camera and the vehicle. This thesis proposes a computer vision based software solution which can estimate a camera’s orientation relative to the movement direction of the vehicle by utilizing video data. The task was solved by using feature-based methods and open source software. When using simulated data sets, the camera orientation estimates had an absolute error of 0.31 degrees on average. The software solution can be integrated to be a part of the traffic sign localization pipeline of the company in question.

Pose estimation of a humanoid robot using images from a mobile external camera

NOGUEIRA, Marcelo B. ; MEDEIROS, Adelardo A. D. ; ALSINA, Pablo J. Pose Estimation of a Humanoid Robot Using Images from an Mobile Extern Camera. In: IFAC WORKSHOP ON MULTIVEHICLE SYSTEMS, 2006, Salvador, BA. Anais... Salvador: MVS 2006, 2006.

Determination of a Mean Solar Radiation Year and of a Typical Meteorological Year for the region of Funchal in the Madeira Island

This paper presents the determination of a mean solar radiation year and of a typical meteorological year for the region of Funchal in the Madeira Island, Portugal. The data set includes hourly mean and extreme values for air temperature, relative humidity and wind speed and hourly mean values for solar global and diffuse radiation for the period 2004-2014, with maximum data coverage of 99.7%. The determination of the mean solar radiation year consisted, in a first step, in the average of all values for each pair hour/day and, in a second step, in the application of a five days centred moving average of hourly values. The determination of the typical meteorological year was based on Finkelstein-Schafer statistics, which allows to obtain a complete year of real measurements through the selection and combination of typical months, preserving the long term averages while still allowing the analysis of short term events. The typical meteorological year validation was carried out through the comparison of the monthly averages for the typical year with the long term monthly averages. The values obtained were very close, so that the typical meteorological year can accurately represent the long term data series. The typical meteorological year can be used in the simulation of renewable energy systems, namely solar energy systems, and for predicting the energy performance of buildings.

Direct determination of Cu, Cd, Ni and Pb in aquatic humic substances by graphite furnace atomic absorption spectrometry

A method has been developed for the direct determination of Cu, Cd, Ni and Pb in aquatic humic substances (AHS) by graphite furnace atomic absorption spectrometry. AHS were isolated from water samples rich in organic matter, collected in the Brazilian Ecological Parks. All analytical curves presented good linear correlation coefficient. The limits of detection and quantification were in the ranges 2.5-16.7 mu g g(-1) and 8.5-50.0 mu g g(-1), respectively. The accuracy was determined using recovery tests, and for all analytes recovery percentages ranged from 93 - 98 %, with a relative standard deviation less than 4 %. The results indicated that the proposed method is a suitable alternative for the direct determination of metals in AHS.

Voltammetric determination of isoprenaline in pharmaceutical preparations using a copper(II) hexacyanoferrate(III) modified carbon paste electrode

The electroanalytical determination of isoprenaline in pharmaceutical preparations of a homemade carbon paste electrode modified with copper(II) hexacyanoferrate(III) (CuHCF) was studied by cyclic voltammetry. Several parameters were studied for the optimization of the sensor such as electrode composition, electrolytic solution, pH effect, potential scan rate and interferences in potential. The optimum conditions were found in an electrode composition (in mass) of 15% CuHCF, 60% graphite and 25% mineral oil in 0.5 mol l(-1) acetate buffer solution at pH 6.0. The analytical curve for isoprenaline was linear in the concentration range from 1.96 x 10(-4) to 1.07 x 10(-3) mol l(-1) with a detection limit of 8.0 x 10(-5) mol l(-1). The relative standard deviation was 1.2% for 1.96 x 10(-4) mol l(-1) isoprenaline solution (n=5). The procedure was successfully applied to the determination of isoprenaline in pharmaceutical preparations; the CuHCF modified carbon paste electrode gave comparable results to those results obtained using a UV spectrophotometric method. (C) 2004 Elsevier B.V. All rights reserved.

Flow-injection spectrophotometric determination of dipyrone in pharmaceutical formulations using ammonium molybdate as chromogenic reagent

A simple and rapid flow-injection spectrophotometric method is reported for the determination of dipyrone in pharmaceutical formulations. The method is based on the reaction of dipyrone with ammonium molybdate in acidic medium to produce blue molybdenum, which was detected spectrophotometrically at 620 nm. The analyte was determined in a single-line flow system. The calibration curve obtained was linear in the range of 5x10(-4) to 8x10(-3) mol L-1 for dipyrone concentration and the precision ( s r =1.7%) was satisfactory. The method proved to be selective and adequately sensitive. Application of the method to the analysis of pharmaceutical samples resulted in excellent accuracy; the percent mean recoveries were in the range 95.3%-101% and relative errors less than 5.0% for five pharmaceutical formulations were found.

Age and growth, mortality, reproduction and relative yield per recruit of the bogue, Boops boops Linne, 1758 (Sparidae), from the Algarve (south of Portugal) longline fishery

Samples of Boops boops ranging from 7.4 to 30.5 cm were obtained mainly by longline, supplemented by beach seining in the Ria Formosa lagoon, and by market sampling in the Algarve (southern Portugal). The macroscopic analyses of the gonads and the gonad somatic index showed that the south coast of Portugal B. boops spawn mainly from late winter to spring, between February and May. The length at first maturity was similar for males and females and the value for both sexes combined was estimated to be 15.22 cm, corresponding to an age range of 1-3. Age was determined by reading growth bands on otoliths. Age determination was validated by marginal increment analysis. The estimated parameters were L-infinity = 28.06, K = 0.22 and t(0) = -1.42. Mortality rates were calculated for fish captured with longlines, and the estimated parameters were M = 0.33, Z = 1.04 and F = 0.71. Relative yield per recruit analysis and sensitivity analysis showed that the resource is moderately exploited. From the perspective of sustainability, these results provide support for the use of longlines as a gear that is among the least harmful for species such as the bogue.

A Highly Improved Method For Sensitive Determination Of Amitriptyline In Pharmaceutical Formulations Using An Unmodified Carbon Nanotube Electrode In The Presence Of Sulfuric Acid.

The present paper describes a novel, simple and reliable differential pulse voltammetric method for determining amitriptyline (AMT) in pharmaceutical formulations. It has been described for many authors that this antidepressant is electrochemically inactive at carbon electrodes. However, the procedure proposed herein consisted in electrochemically oxidizing AMT at an unmodified carbon nanotube paste electrode in the presence of 0.1 mol L(-1) sulfuric acid used as electrolyte. At such concentration, the acid facilitated the AMT electroxidation through one-electron transfer at 1.33 V vs. Ag/AgCl, as observed by the augmentation of peak current. Concerning optimized conditions (modulation time 5 ms, scan rate 90 mV s(-1), and pulse amplitude 120 mV) a linear calibration curve was constructed in the range of 0.0-30.0 μmol L(-1), with a correlation coefficient of 0.9991 and a limit of detection of 1.61 μmol L(-1). The procedure was successfully validated for intra- and inter-day precision and accuracy. Moreover, its feasibility was assessed through analysis of commercial pharmaceutical formulations and it has been compared to the UV-vis spectrophotometric method used as standard analytical technique recommended by the Brazilian Pharmacopoeia.

Development And Shelf-life Determination Of Pasteurized, Microfiltered, Lactose Hydrolyzed Skim Milk.

The segment of the world population showing permanent or temporary lactose intolerance is quite significant. Because milk is a widely consumed food with an high nutritional value, technological alternatives have been sought to overcome this dilemma. Microfiltration combined with pasteurization can not only extend the shelf life of milk but can also maintain the sensory, functional, and nutritional properties of the product. This studied developed a pasteurized, microfiltered, lactose hydrolyzed (delactosed) skim milk (PMLHSM). Hydrolysis was performed using β-galactosidase at a concentration of 0.4mL/L and incubation for approximately 21h at 10±1°C. During these procedures, the degree of hydrolysis obtained (>90%) was accompanied by evaluation of freezing point depression, and the remaining quantity of lactose was confirmed by HPLC. Milk was processed using a microfiltration pilot unit equipped with uniform transmembrane pressure (UTP) ceramic membranes with a mean pore size of 1.4 μm and UTP of 60 kPa. The product was submitted to physicochemical, microbiological, and sensory evaluations, and its shelf life was estimated. Microfiltration reduced the aerobic mesophilic count by more than 4 log cycles. We were able to produce high-quality PMLHSM with a shelf life of 21 to 27d when stored at 5±1°C in terms of sensory analysis and proteolysis index and a shelf life of 50d in regard to total aerobic mesophile count and titratable acidity.