Proposed mechanism for the selection of lactase persistence in childhood.

Lactase persistence/persistent (LP), the ability to express the lactase enzyme in adults, is one of the most strongly selected phenotypes in humans. It is encoded by at least five genetic variants that have rapidly become widespread in various human populations. The underlying selective mechanism is not clear however, because dairy products in general are well tolerated in adults, even by lactase non-persistence/persistent (LNP) individuals. Cultural adaptations to milk consumption, notably fermentation and transformation, which can provide most of the energy (protein, fat) to both LP and LNP individuals without any associated cost seem to have been common in ancient societies. Here, we propose that selection for LP occurred through increased glucose/galactose (energy) from fresh milk intake in early childhood, a crucial period for growth. At the age of weaning indeed, lactase activity has already begun to decline in LNP individuals so the gain in energy from fresh milk by LP children represents a major fitness increase.

CASD-NMR 2: robust and accurate unsupervised analysis of raw NOESY spectra and protein structure determination with UNIO.

UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved--including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO-ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications.

Rapid proton-detected NMR assignment for proteins with fast magic angle spinning.

Using a set of six (1)H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5-30 kDa proteins. The approach relies on perdeuteration, amide (2)H/(1)H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary (13)C/(15)N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Advances in automated NMR protein structure determination.

Around half of all protein structures solved nowadays using solution-state nuclear magnetic resonance (NMR) spectroscopy have been because of automated data analysis. The pervasiveness of computational approaches in general hides, however, a more nuanced view in which the full variety and richness of the field appears. This review is structured around a comparison of methods associated with three NMR observables: classical nuclear Overhauser effect (NOE) constraint gathering in contrast with more recent chemical shift and residual dipole coupling (RDC) based protocols. In each case, the emphasis is placed on the latest research, covering mainly the past 5 years. By describing both general concepts and representative programs, the objective is to map out a field in which--through the very profusion of approaches--it is all too easy to lose one's bearings.

Mapping protein conformational energy landscapes using NMR and molecular simulation.

Nuclear magnetic resonance (NMR) spectroscopy provides detailed understanding of the nature and extent of protein dynamics on physiologically important timescales. We present recent advances in the combination of NMR with state-of-the-art molecular simulation that are providing unique new insight into the motions on timescales from nanoseconds to milliseconds. In particular, we focus on methods based on residual dipolar couplings (RDCs) that allow for detailed mapping of the protein conformational energy landscape. A novel combination of RDCs with accelerated molecular dynamics allows for the development of ensemble representations of the underlying Boltzmann ensemble.

31P MAS refocused INADEQUATE spin-echo (REINE) NMR spectroscopy: revealing J coupling and chemical shift two-dimensional correlations in disordered solids.

Two-dimensional (2D) variations in (2)J(P(1),P(1)), (2)J(P(1),P(2)), and (2)J(P(2),P(2)) are obtained--using the REINE (REfocused INADEQUATE spin-Echo) pulse sequence presented by Cadars et al. (Phys. Chem. Chem. Phys. 2007, 9, 92-103)--from pixel-by-pixel fittings of the spin-echo modulation for the 2D correlation peaks due to linked phosphate tetrahedra (P(1)-P(1), P(1)-P(2), P(2)-P(1), and P(2)-P(2)) in a (31)P refocused INADEQUATE solid-state MAS NMR spectrum of a cadmium phosphate glass, 0.575CdO-0.425P(2)O(5). In particular, separate variations for each 2D (31)P REINE peak are obtained which reveal correlations between the J couplings and the (31)P chemical shifts of the coupled nuclei that are much clearer than those evident in previously presented 2D z-filtered (31)P spin-echo spectra. Notably, such correlations between the J couplings and the (31)P chemical shifts are observed even though the conditional probability distributions extracted using the protocol of Cadars et al. (J. Am. Chem. Soc. 2005, 127, 4466-4476) indicate that there is no marked correlation between the (31)P chemical shifts of neighboring phosphate tetrahedra. For 2D peaks at the P(2) (31)P chemical shift in the direct dimension, there can be contributions from chains of three units (P(1)-P(2)-P(1)), chains of four units (P(1)-P(2)-P(2)-P(1)), or longer chains or rings (-P(2)-P(2)-P(2)-): for the representative glass considered here, best fits are obtained assuming a glass comprised predominantly of chains of four units. The following variations are found: (2)J(P(1),P(1)) = 13.4 +/- 0.3 to 14.8 +/- 0.5 Hz, (2)J(P(1),P(2)) = 15.0 +/- 0.3 to 18.2 +/- 0.3 Hz, and (2)J(P(2),P(2)) = 5.9 +/- 0.6 to 9.1 +/- 0.9 Hz from the fits to the P(1)-P(1), P(1)-P(2), and P(2)-P(2) peaks, respectively. The correlation of a particular J coupling with the (31)P chemical shifts of the considered nucleus and the coupled nucleus is quantified by the coefficients C(F(2)) and C(F(1)) that correspond to the average pixel-by-pixel change in the J coupling with respect to the chemical shift of the observed (F(2)) and neighboring (F(1)) (31)P nuclei, respectively.

A multi-substrate screening approach for the identification of a broadly applicable Diels-Alder catalyst.

When developing a synthetic methodology, chemists generally optimize a single substrate and then explore the substrate scope of their method. This approach has led to innumerable and widely-used chemical reactions. However, it frequently provides methods that only work on model substrate-like compounds. Perhaps worse, reaction conditions that would enable the conversion of other substrates may be missed. We now show that a different approach, originally proposed by Kagan, in which a collection of structurally distinct substrates are evaluated in a single reaction vessel, can not only provide information on the substrate scope at a much earlier stage in methodology development, but even lead to a broadly applicable synthetic methodology. Using this multi-substrate screening approach, we have identified an efficient and stereoselective imidodiphosphorimidate organocatalyst for scalable Diels-Alder reactions of cyclopentadiene with different classes of α,ß-unsaturated aldehydes.

Strong-coupling induced damping of spin-echo modulations in magic-angle-spinning NMR: Implications for J coupling measurements in disordered solids.

In the context of improving J coupling measurements in disordered solids, strong coupling effects have been investigated in the spin-echo and refocused INADEQUATE spin-echo (REINE) modulations of three- and four-spin systems under magic-angle-spinning (MAS), using density matrix simulations and solid-state NMR experiments on a cadmium phosphate glass. Analytical models are developed for the different modulation regimes, which are shown to be distinguishable in practice using Akaike's information criterion. REINE modulations are shown to be free of the damping that occurs for spin-echo modulations when the observed spin has the same isotropic chemical shift as its neighbour. Damping also occurs when the observed spin is bonded to a strongly-coupled pair. For mid-chain units, the presence of both direct and relayed damping makes both REINE and spin-echo modulations impossible to interpret quantitatively. We nonetheless outline how a qualitative comparison of the modulation curves can provide valuable information on disordered networks, possibly also pertaining to dynamic effects therein.

Comprehensive automation for NMR structure determination of proteins.

This chapter gives an overview of automated protein structure determination by nuclear magnetic resonance (NMR) with the UNIO protocol that enables high to full automation of all NMR data analysis steps involved. Four established algorithms, namely, the MATCH algorithm for sequence-specific resonance assignment, the ASCAN algorithm for side-chain resonance assignment, the CANDID algorithm for NOE assignment, and the ATNOS algorithm for signal identification in NMR spectra, are assembled into three principal UNIO NMR data analysis components (MATCH, ATNOS/ASCAN, and ATNOS/CANDID) that are accessed thanks to a particularly intuitive and flexible, yet powerful graphical user interface (GUI). UNIO is designed to work independently or in association with other NMR software. The principal data analysis components for sequence-specific backbone, side-chain and NOE assignment may be run separately or out of sequence. User-intervention at individual stages is encouraged and facilitated by graphical tools included for the preparation, analysis, validation, and subsequent presentation of the NMR structure.

Blind testing of routine, fully automated determination of protein structures from NMR data.

The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.

Ti K-edge XANES study of the local environment of titanium in bioresorbable TiO2-CaO-Na2O-P2O5 glasses.

Ti K-edge XANES (X-ray absorption near edge structure) spectroscopy has been used to study the local coordination of titanium in biocompatible and bioresorbable TiO2-CaO-Na2O-P2O5 glasses. Both conventional melt-quenched glasses of composition (TiO2)x(CaO)0.30(Na2O)0.20-x(P2O5)0.50, where x = 0.01, 0.03 and 0.05, and sol-gel derived (TiO2)0.25(CaO)0.25(P2O5)0.50 glass have been studied. The results show that in all the materials studied, titanium is surrounded by an octahedron of oxygen atoms. Further analysis reveals that the TiO6 site in the amorphous samples is not heavily distorted relative to that in rutile, anatase or CaSiTiO5. The spectra from the (TiO2)0.25(CaO)0.25(P2O5)0.50 sol-gel samples reveal greater distortion in the TiO6 site in the dried gel compared to the heat-treated sol-gel glass. The XANES spectra from melt-quenched glass samples soaked in distilled water for various times do not shown any evidence of degradation of the titanium site over periods of up to 14 days.

Breast cancer care: changing community standards.

A community hospital-based program was developed to improve breast cancer care in the community. A consensus was developed for what should be optimal care; a database was established to document the care being delivered in the community; and the data were analyzed to document changes in practice patterns over time. The major clinical benefits to patients included a significant improvement in needle biopsy rates, decreased utilization of second operative procedures, increased breast conservation surgery, conformity to guidelines for adjuvant chemotherapy administration, and a sizable increase in discovery of small breast cancers by screening mammography.

The use of FISH on breast core needle samples for the presurgical assessment of HER-2 oncogene status.

HER-2 status has been used in breast carcinoma as a prognostic marker to predict drug response and to select patients for trastuzumab treatment. Since immunohistochemistry (IHC) is thought to be less reliable, HER-2 testing with FISH is preferred. The analysis of HER-2 is usually performed on formalin-fixed paraffin tissue sections obtained from surgery. The use of paraffin sections is very time consuming and labor intensive. The objectives of this study were to (1) develop a simple and quick FISH protocol using touch imprints of breast core needle biopsies, eliminating the deparaffinization and pretreatment; and (2) make the HER-2 status available at the presurgical multidisciplinary treatment planning conference. A total of 50 core samples of breast carcinoma were obtained from image-guided core needle biopsy. Both FISH and IHC data were available for 46 cases. Forty-four of 46 cases (95.7%) were consistent. Two IHC 2+ cases were nonamplified (ratios of 0.99 and 1.09). It is expected that, in the near future, additional molecular markers will be used before surgery when the overall treatment plan is being developed. We conclude that HER-2 gene analysis by FISH on breast touch imprints is easily done and is a useful and reliable technique.