Nutritional epidemiology of postmenopausal breast cancer in western New York

The authors studied 439 postmenopausal breast cancer cases, identified in hospitals throughout western New York, with an interview schedule that considered frequency and amount ingested of 172 foods and provided data for an estimate of total calories ingested. These were compared with age-matched controls comprising a random sample of the same communities as the cases. The extensive interviews, requiring 2.0 hours on average to administer, also covered alcohol ingestion, Quetelet index, and a wide variety of reproductive factors. The authors found, as have most investigators over the past 25 years, that risk increased with increases in age at first pregnancy, decreased with increases in numbers of children and pregnancies, and increased in those with history of benign breast disease and in those with female relatives previously affected with breast cancer. Risk adjusted for potential confounders was highest among women with the lowest ingestion of carotene or a substance correlated with its ingestion. Risk was not associated with retinol ingestion. It increased with increases in Quetelet index. Fat intake, whether studied in terms of quantity or the proportion of total calories derived from fat, was not associated with risk of breast cancer. Our analyses of these factors were adjusted for age, education, and the reproductive history traits described above.     

Occupational risk factors for small bowel carcinoid tumor: a European population-based case-control study

Small bowel carcinoid tumor (SBC) is a rare disease of unknown etiology but with an age-, sex-, and place-specific occurrence that may indicate an occupational origin. A European multicenter population-based case-control study was conducted from 1995 through 1997. Incident SBC cases between 35 and 69 years of age (n = 101) were identified, together with 3335 controls sampled from the catchment area of the cases. Histological review performed by a reference pathologist left 99 cases for study; 84 cases and 2070 population controls were interviewed. The industries most closely associated (a twofold or more odds ratio [OR]) with SBC, taking into account a 10-year time lag after exposure were, among women, employment in wholesale industry of food and beverages (OR, 8.2; 95% confidence interval [CI], 1.9 to 34.9]) and among men, manufacture of motor vehicle bodies (OR, 5.2; 95% CI, 1.2 to 22.4), footwear (OR, 3.9; 95% CI, 0.9 to 16.1), and metal structures (OR, 3.3; 95% CI, 1.0 to 10.4). The identified high-risk occupations with an OR above 2 were shoemakers, structural metal preparers, construction painters and other construction workers, bookkeepers, machine fitters, and welders (men). The OR for regular occupational use of organic solvents for at least half a year was 2.0 (95% CI, 1.0 to 4.2). Exposure to rust-preventive paint containing lead was suggested as another potential occupational exposure (OR, 9.1; 95% CI, 0.8 to 107). This explorative study suggests an association between certain occupational exposures and SBC, but some of these associations could be attributable to chance. All findings should be regarded as tentative.     

A comparison between the use of dynamic mechanical analysis and oscillatory shear rheometry for the characterisation of hydrogels

In this study the use of dynamic mechanical analysis (DMA) for the mechanical characterisation of pharmaceutical hydrogels was evaluated. DMA was used in two different modes, the "controlled force" and the "multi-strain" (MS). The results obtained on dextran methacrylate hydrogels of various compositions were compared to those obtained using an oscillatory shear rheometer. The best agreement was found between the MS-DMA and the rheometer results. The moduli measured in MS-DMA were extrapolated towards zero compression to obtain the modulus of the hydrogels. This procedure resulted in good agreement with the data obtained with the rheometer. Hydrogels were analysed after swelling to equilibrium with both methods, DMA and rheology. A scaling between the elastic modulus (G') and the equilibrium swollen polymer volume fraction (v(2,s)) could be found, although the best correlation between G' and v(2,s) was obtained with the rheometer.     

Statistical analysis of ENDOR spectra

Electron–nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the Y122• in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with ¹H/²H isotopic labeling of Y122• in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Structural information at atomic resolution is essential for many areas of physics, chemistry, and biology. Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) (1) belongs to a pool of methods, including nuclear magnetic resonance, optical spectroscopy, X-ray diffraction, and cryo-electron microscopy, which can deliver this information. For biophysical applications, the EPR-based electron–nuclear double resonance (ENDOR) technique is particularly suited for mechanistic studies involving endogenous paramagnetic centers, such as redox-active amino acid radical intermediates or metal ions and clusters, as it measures the hyperfine (hf) interaction between these centers and their surrounding magnetic nuclei (2), providing information on the protein architecture and establishing structural to functional relationships. The method has identified and characterized a wealth of paramagnetic intermediates in enzymes; for recent examples see refs. 3–7. Moreover, ENDOR spectroscopy has recently been proposed as a method of choice to measure molecular distances in the angstrom to nanometer range by ¹⁹F spin labeling (8), bridging a critical gap in pulse EPR-based distance measurements in biomolecules and complementing ¹⁹F-based NMR spectroscopy (9, 10).ENDOR spectra of magnetic nuclei are usually recorded in the solid state (a frozen solution of a biological sample) at very low temperatures (T < 80 K) and thus display the full anisotropy of magnetic interactions, resulting in very broad lines. The signals are recorded as a change of the EPR electron-spin echo (11), which is sensitive to drifts in experimental conditions, particularly at low temperatures. Therefore, a model-free analysis of the observed signals constitutes one of the main challenges of ENDOR spectroscopy. In the last decade, progress in EPR instrumentation enabled the implementation of pulsed EPR spectrometers operating in the quasi-optical regime (12). We have recently reported a ¹H ENDOR study of the essential Y122• in the wild-type (WT) β2 subunit of Escherichia coli ribonucleotide reductase (RNR) (Fig. 1A), using a commercial instrument (13) operating at 9.4 T/263 GHz. The strong orientation selection (hole burning) under high field/frequency conditions leads to sharpening of the spectra, strongly facilitating their analysis. We demonstrated the effect with the ¹H spectrum of Y122•, illustrated in Fig. 1B, where the sharp peaks were attributed to internal ¹Hs of the radical. Additional broad resonances became visible, whose attribution presents additional challenges. From visual inspection alone, the significance of these signals could not be inferred given their shallow line shapes and the difficulty to separate them from unknown baseline distortions.Open in a separate windowFig. 1.(A) Structure of Y122 and surrounding amino acids in the E. coli RNR WT-β2 subunit from PDB 1mxr (22) in the reduced state (phenolic proton omitted). Highlighted are interactions with two protons at distances <3 Å from the tyrosine oxygen. Protons were added with Pymol 2.2.2 in positions determined by the overall orientation of the amino acids. The O-O distance (3.8 Å) to the water molecule coordinating the proximal Fe ion is highlighted as well. (B) The 263-GHz ¹H Davies ENDOR spectrum of Y122• adapted from ref. 13. Marked are resonances of the ring protons. Unknown broad features (asterisks) became observable only at 263 GHz. (Inset) Structure of a Y• with labeling of the internal protons.The structure and function of amino acid radicals, particularly Y• in prototype biological machineries such as RNRs or photosystem II, have been the focus of several studies since the 1990s, due to their representative role in biological redox reactions and proton-coupled electron transfer (14, 15). In class Ia RNRs, the Y•/diiron cofactor is essential for enzyme activity and thus for cell survival (16). In contrast to nonprotein Y• model systems (17, 18), the conformation of Y•s in proteins appeared much more constrained (19, 20). This finding was rationalized as due to the interaction with the protein environment that confers redox properties and thus governs biological function. Nevertheless, the mechanism of action of Y• at the diiron cluster in class Ia RNRs remains puzzling. The current model for initiating nucleotide reduction requires that Y122• (E. coli numbering) is reduced in combination with protonation by a water molecule bound to the diferric cluster (16, 21) when the RNR subunits β2, α2 are mixed with substrate and allosteric effector. In all structures of β2 alone, however, the distance between the oxygen of the water and the oxygen of Y122• is too long for this step [3.8 Å in Protein Data Bank (PDB) 1mxr (22); Fig. 1A]. Thus, the model requires a conformational change to shorten this distance, for which to date no experimental evidence exists.Statistical methods are valuable to support the interpretation of biophysical experiments (23, 24). They have already been introduced in EPR spectroscopy for the analysis of distance measurements and distance distributions (25, 26). While adding uncertainty quantification to general spectroscopic practice, these works still hinge on assumptions about functions of interest, whether explicitly made through a Bayesian prior distribution or implicitly included in a choice of a penalization functional or thresholding procedure, some of which are hard to verify in practice. Additionally, ENDOR targets a different object of interest as it uses a different physical method. Therefore, a statistical analysis of ENDOR signals requires different methodology and has been less explored, possibly also due to a high incidence of systematic errors that are hard to model. In this contribution we aim at a statistical model that does not hinge on any specific assumptions regarding the functions of interest, in particular not on any that are hard to verify. Hence, our statistical model can be generalized to other biophysical experiments. In particular, our statistical approach includes careful statistical checking of distributional assumptions and thus lays a strong foundation for our statistical hypothesis tests. Recent advances in microwave (mw) technology, which allow for long-term signal stability and thus accumulation of large datasets, now provide the opportunity to gain additional information from quantitative ENDOR analysis. In this context, development of statistical methods that disentangle signals from noise and systematic error without having to rely on any assumptions regarding the spectrum and that report uncertainty estimates becomes mandatory.The topic of this paper is a statistical analysis of ENDOR spectra and its representative application to assess the significance of broad spectral features observed in the 263-GHz ¹H spectra of the E. coli RNR Y122•. We present a statistical model for the experimental data, which is used to extract the “most likely signal.” This model takes advantage of the information hidden in each individual scan (or batch) of the spectrum that is usually lost in the process of signal averaging. The treatment presents estimation of the uncertainty in the ENDOR spectra and permits subsequent statistical tests. The mathematical approach combined with spectroscopy of various isotopically labeled mutants of Y122• ultimately uncovered a distribution of conformations of Y122•, yielding insight into the mechanism of action of this essential radical.     

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state ³[Re ]^∗ is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.