Re-examining the spectra of macromolecules. Current practice of spectral quasi B-factor flattening

The analysis of structure factors in 3D cryo-EM Coulomb potential maps and their “enhancement” at the end of the reconstruction process is a well-established practice, normally referred to as sharpening. The aim is to increase contrast and, in this way, to help tracing the atomic model. The most common way to accomplish this enhancement is by means of the so-called B-factor correction, which applies a global filter to boost high frequencies with some dampening considerations related to noise amplification. The results are maps with a better visual aspect and a quasiflat spectrum at medium and high frequencies. This practice is so widespread that most map depositions in the Electron Microscopy Data Base (EMDB) only contain sharpened maps. Here, the use in cryoEM of global B-factor corrections is theoretically and experimentally analyzed. Results clearly illustrate that protein spectra present a falloff. Thus, spectral quasi-flattening may produce protein spectra with distortions when compared with experimental ones, this fact, combined with the practice of reporting only sharpened maps, generates a sub-optimal situation in terms of data preservation, reuse and reproducibility. Now that the field is more advanced, we put forward two suggestions: (1) to use methods which keep more faithfully the original experimental signal properties of macromolecules when “enhancing” the map, and (2) to further stress the need to deposit the original experimental maps without any postprocessing or sharpening, not only the enhanced maps. In the absence of access to these original maps data is lost, preventing their future analysis with new methods.     

Use of the twin-cell differential titration calorimeter for binding studies. I. EDTA and its calcium complex

The use of a twin-cell differential titration calorimeter is described which utilizes small volumes (1–3 ml) of modest concentrations of materials (0.001–0.01 M) and yield data of good precision. Operation is controlled by a microprocessor which regulates and controls the addition of reagents and collects and displays the data as time, temperature in volts, and the pH. Corrections for the titration of water are applied to the potentiometric data, and the thermal data are corrected for the initial temperature-time baseline, the changes in heat capacity, and the heat loss (or gain) to the external environment. Finally, the thermal signal is corrected for the heat derived from the formation of water due to the free hydrogen or hydroxyl ions present. The corrected data as pH, groups titrated adn ΔH_T (kcal/mol) can then be used to obtain the parameters pK′ and ΔH_i involved with the equilibria by curve-fitting the observed data.

The system has been applied to the ionization of EDTA and its calcium complex. The ionization constants, the heats of ionization, the stoichiometry of binding and the heat of binding have been determined and demonstrated to be in agreement with published values.     

Correcting Coalescent Analyses for Panel-Based SNP Ascertainment

Single-nucleotide polymorphism (SNP) data are routinely obtained by sequencing a region of interest in a small panel, constructing a chip with probes specific to sites found to vary in the panel, and using the chip to assay subsequent samples. The size of the chip is often reduced by removing low-frequency alleles from the set of SNPs. Using coalescent estimation of the scaled population size parameter, Θ, as a test case, we demonstrate the loss of information inherent in this procedure and develop corrections for coalescent analysis of SNPs obtained via a panel. We show that more accurate Θ-estimates can be recovered if the panel size is known, but at considerable computational cost as the panel individuals must be explicitly modeled in the analysis. We extend this technique to apply to the case where rare alleles have been omitted from the SNP panel. We find that when appropriate corrections for panel ascertainment and rare-allele omission are used, the biases introduced by ascertainment are largely correctable, but recovered estimates are less accurate than would be obtained with fully sequenced data. This method is then applied to recombinant multiple population data to investigate the effects of recombination and migration on the estimate of Θ.     

Transient binding of plastocyanin to its physiological redox partners modifies the copper site geometry

The transient complexes of plastocyanin with cytochrome f and photosystem I are herein used as excellent model systems to investigate how the metal sites adapt to the changes in the protein matrix in transient complexes that are involved in redox reactions. Thus, both complexes from the cyanobacterium Nostoc sp. PCC 7119 (former Anabaena sp. PCC 7119) have been analysed by X-ray absorption spectroscopy. Our data are consistent with a significant distortion of the trigonal pyramidal geometry of the Cu coordination sphere when plastocyanin binds to cytochrome f, no matter their redox states are. The resulting tetrahedral geometry shows a shortening of the distance between Cu and the S(delta) atom of its ligand Met-97, with respect to the crystallographic structure of free plastocyanin. On the other hand, when plastocyanin binds to photosystem I instead of cytochrome f, the geometric changes are not significant but a displacement in charge distribution around the metal centre can be observed. Noteworthy, the electronic density around the Cu atom increases or decreases when oxidised plastocyanin binds to cytochrome f or photosystem I, respectively, thus indicating that the protein matrix affects the electron transfer between the two partners during their transient interaction.     

Pièges,précautions et artefacts en scintigraphie myocardique : illustrations sur caméras semi-conducteurs

Since many years, the myocardial perfusion scintigraphy plays a leading role in the management of coronary artery disease because of its excellent performance for the diagnosis of myocardial ischemia. This exam has also a value widely recognized for risk stratification, assessment of the effectiveness of treatments and research of myocardial viability. The realization of this review is composite with many steps and persons which can potentially provide artefacts. The advent of semiconductor cameras significantly reduce some artefacts, but was not accompanied by a complete removal of these latters, which for some of them may be exacerbated. The use of such cameras will therefore require a learning curve to understand these evolutions. In this article, we propose a clinical illustration of centering artefacts specific of semiconductor cameras, examples of motion artefacts (respiratory and patient motion) and apical artefacts. These abnormalities are commonly encountered during daily practice of myocardial scintigraphy.     

Considerations for normalization of DNA methylation data by Illumina 450K BeadChip assay in population studies

Analysis of epigenetic mechanisms, particularly DNA methylation, is of increasing interest for epidemiologic studies examining disease etiology and impacts of environmental exposures. The Infinium HumanMethylation450 BeadChip^® (450K), which interrogates over 480?000 CpG sites and is relatively cost effective, has become a popular tool to characterize the DNA methylome. For large-scale studies, minimizing technical variability and potential bias is paramount. The goal of this paper was to evaluate the performance of several existing and novel color channel normalizations designed to reduce technical variability and batch effects in 450K analysis from a large population study. Comparative assessment of 10 normalization procedures included the GenomeStudio^® Illumina procedure, the lumi smooth quantile approach, and the newly proposed All Sample Mean Normalization (ASMN). We also examined the performance of normalizations in combination with correction for the two types of Infinium chemistry utilized on the 450K array. We observed that the performance of the GenomeStudio^® normalization procedure was highly variable and dependent on the quality of the first sample analyzed in an experiment, which is used as a reference in this procedure. While the lumi normalization was able to decrease batch variability, it increased variation among technical replicates, potentially reducing biologically meaningful findings. The proposed ASMN procedure performed consistently well, both at reducing batch effects and improving replicate comparability. In summary, the ASMN procedure can improve existing color channel normalization, especially for large epidemiologic studies, and can be successfully implemented to enhance a 450K DNA methylation data pipeline.     

PARP-mediated repair,homologous recombination,and back-up non-homologous end joining-like repair of single-strand nicks

Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed ‘nickase’ variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.