Image_1_A prospective investigation of the effects of soccer heading on cognitive and sensorimotor performances in semi-professional female players.jpg

IntroductionRepetitive head impacts (RHI) from routine soccer (football) heading have been suggested to contribute to the long-term development of neurodegenerative disorders. However, scientific evidence concerning the actual risk of these RHI on brain health remains inconclusive. Moreover, female athletes—despite a presumably increased vulnerability toward the effects of RHI—are largely underrepresented in previous approaches. Therefore, our aim was to prospectively investigate the effects of heading on cognitive and sensorimotor performances, health perception, and concussion symptoms in semi-professional female soccer players.MethodsAn extensive test battery was used to assess cognitive and sensorimotor performances as well as health status (SF-36) and concussion symptoms (SCAT3) of a total of 27 female soccer players (22.2 ± 4.2 years) and 15 control subjects (23.2 ± 3.0 years) before and after one-and-a-half years. Throughout this period, soccer players’ heading exposure was determined using video analysis.ResultsSubgroup comparisons (control [n = 12], low exposure [n = 7], high exposure [n = 8]) showed no time-dependent differences in SF-36 or SCAT3 scores. Similarly, across most behavioral tests, soccer players’ performances evolved equally or more favorably as compared to the control subjects. However, there were significant effects pointing toward slightly negative consequences of heading on aspects of fine motor control (p = 0.001), which were confirmed by correlation and multiple regression analyses. The latter, further, yielded indications for a relationship between heading exposure and negative alterations in postural control (p = 0.002).DiscussionOur findings do not provide evidence for negative effects of soccer heading on female players’ health perception, concussion symptoms, and cognitive performances over the course of one-and-a-half years. However, we found subtle negative alterations in fine motor and postural control that could be attributed to heading exposure. Other factors, like the number of previous head injuries, were not linked to the observed changes. Given the reduction of our initial sample size due to player fluctuation, the results need to be interpreted with caution and validated in larger-scale studies. These should not only focus on cognitive outcomes but also consider sensorimotor changes as a result of RHI from soccer heading.</p

Optimizing Crystal Size of Photosystem II by Macroseeding: Toward Neutron Protein Crystallography

Photosystem II (PSII) catalyzes the photo-oxidation of water to molecular oxygen and protons. The water splitting reaction occurs inside the oxygen-evolving complex (OEC) via a Mn₄CaO₅ cluster. To elucidate the reaction mechanism, detailed structural information for each intermediate state of the OEC is required. Despite the current high-resolution crystal structure of PSII at 1.85 Å and other efforts to follow the structural changes of the Mn₄CaO₅ cluster using X-ray free electron laser (XFEL) crystallography in addition to spectroscopic methods, many details about the reaction mechanism and conformational changes in the catalytic site during water oxidation still remain elusive. In this study, we present a rarely found successful application of the conventional macroseeding method to a large membrane protein like the dimeric PSII core complex (dPSIIcc). Combining microseeding with macroseeding crystallization techniques allowed us to reproducibly grow large dPSIIcc crystals with a size of ∼3 mm. These large crystals will help improve the data collected from spectroscopic methods like polarized extended X-ray absorption fine structure (EXAFS) and single crystal electron paramagnetic resonance (EPR) techniques and are a prerequisite for determining a three-dimensional structure using neutron diffraction

The Protonation States of Oxo-Bridged Mn^IV Dimers Resolved by Experimental and Computational Mn K Pre-Edge X‑ray Absorption Spectroscopy

In nature, the protonation of oxo bridges is a commonly encountered mechanism for fine-tuning chemical properties and reaction pathways. Often, however, the protonation states are difficult to establish experimentally. This is of particular importance in the oxygen evolving complex of photosystem II, where identification of the bridging oxo protonation states is one of the essential requirements toward unraveling the mechanism. In order to establish a combined experimental and theoretical protocol for the determination of protonation states, we have systematically investigated a series of Mn model complexes by Mn K pre-edge X-ray absorption spectroscopy. An ideal test case for selective bis-μ-oxo-bridge protonation in a Mn dimer is represented by the system [Mn^IV₂(salpn)₂(μ-OH_<i>n</i>)₂]^<i>n</i>+. Although the three species [Mn^IV₂(salpn)₂(μ-O)₂], [Mn^IV₂(salpn)₂(μ-O)­(μ-OH)]⁺ and [Mn^IV₂(salpn)₂(μ-OH)₂]²⁺ differ only in the protonation of the oxo bridges, they exhibit distinct differences in the pre-edge region while maintaining the same edge energy. The experimental spectra are correlated in detail to theoretically calculated spectra. A time-dependent density functional theory approach for calculating the pre-edge spectra of molecules with multiple metal centers is presented, using both high spin (HS) and broken symmetry (BS) electronic structure solutions. The most intense pre-edge transitions correspond to an excitation of the Mn 1s core electrons into the unoccupied orbitals of local e_g character (d_<i>z</i>² and d_<i>xy</i> based in the chosen coordinate system). The lowest energy experimental feature is dominated by excitations of 1s-α electrons, and the second observed feature is primarily attributed to 1s-β electron excitations. The observed energetic separation is due to spin polarization effects in spin-unrestricted density functional theory and models final state multiplet effects. The effects of spin polarization on the calculated Mn K pre-edge spectra, in both the HS and BS solutions, are discussed in terms of the strength of the antiferromagnetic coupling and associated changes in the covalency of Mn–O bonds. The information presented in this paper is complemented with the X-ray emission spectra of the same compounds published in an accompanying paper. Taken together, the two studies provide the foundation for a better understanding of the X-ray spectroscopic data of the oxygen evolving complex (OEC) in photosystem II

X‑ray Emission Spectroscopy as an <i>in Situ</i> Diagnostic Tool for X‑ray Crystallography of Metalloproteins Using an X‑ray Free-Electron Laser

Serial femtosecond crystallography (SFX) using the ultrashort X-ray pulses from a X-ray free-electron laser (XFEL) provides a new way of collecting structural data at room temperature that allows for following the reaction in real time after initiation. XFEL experiments are conducted in a shot-by-shot mode as the sample is destroyed and replenished after each X-ray pulse, and therefore, monitoring and controlling the data quality by using <i>in situ</i> diagnostic tools is critical. To study metalloenzymes, we developed the use of simultaneous collection of X-ray diffraction of crystals along with X-ray emission spectroscopy (XES) data that is used as a diagnostic tool for crystallography, by monitoring the chemical state of the metal catalytic center. We have optimized data analysis methods and sample delivery techniques for fast and active feedback to ensure the quality of each batch of samples and the turnover of the catalytic reaction caused by reaction triggering methods. Here, we describe this active <i>in situ</i> feedback system using Photosystem II as an example that catalyzes the oxidation of H₂O to O₂ at the Mn₄CaO₅ active site. We used the first moments of the Mn Kβ_1,3 emission spectra, which are sensitive to the oxidation state of Mn, as the primary diagnostics. This approach is applicable to different metalloproteins to determine the integrity of samples and follow changes in the chemical states of the reaction that can be initiated by light or activated by substrates and offers a metric for determining the diffraction images that are used for the final data sets

Experimental and Computational X‑ray Emission Spectroscopy as a Direct Probe of Protonation States in Oxo-Bridged Mn^IV Dimers Relevant to Redox-Active Metalloproteins

The protonation state of oxo bridges in nature is of profound importance for a variety of enzymes, including the Mn₄CaO₅ cluster of photosystem II and the Mn₂O₂ cluster in Mn catalase. A set of dinuclear bis-μ-oxo-bridged Mn^IV complexes in different protonation states was studied by Kβ emission spectroscopy to form the foundation for unraveling the protonation states in the native complex. The valence-to-core regions (valence-to-core XES) of the spectra show significant changes in intensity and peak position upon protonation. DFT calculations were performed to simulate the valence-to-core XES spectra and to assign the spectral features to specific transitions. The Kβ_2,5 peaks arise primarily from the ligand 2p to Mn 1s transitions, with a characteristic low energy shoulder appearing upon oxo-bridge protonation. The satellite Kβ″ peak provides a more direct signature of the protonation state change, since the transitions originating from the 2s orbitals of protonated and unprotonated μ-oxo bridges dominate this spectral region. The energies of the Kβ″ features differ by ∼3 eV and thus are well resolved in the experimental spectra. Additionally, our work explores the chemical resolution limits of the method, namely, whether a mixed (μ-O)­(μ-OH₂) motif can be distinguished from a symmetric (μ-OH)₂ one. The results reported here highlight the sensitivity of Kβ valence-to-core XES to single protonation state changes of bridging ligands, and form the basis for further studies of oxo-bridged polymetallic complexes and metalloenzyme active sites. In a complementary paper, the results from X-ray absorption spectroscopy of the same Mn^IV dimer series are discussed

L‑Edge X‑ray Absorption Spectroscopy of Dilute Systems Relevant to Metalloproteins Using an X‑ray Free-Electron Laser

L-edge spectroscopy of 3d transition metals provides important electronic structure information and has been used in many fields. However, the use of this method for studying dilute aqueous systems, such as metalloenzymes, has not been prevalent because of severe radiation damage and the lack of suitable detection systems. Here we present spectra from a dilute Mn aqueous solution using a high-transmission zone-plate spectrometer at the Linac Coherent Light Source (LCLS). The spectrometer has been optimized for discriminating the Mn L-edge signal from the overwhelming O K-edge background that arises from water and protein itself, and the ultrashort LCLS X-ray pulses can outrun X-ray induced damage. We show that the deviations of the partial-fluorescence yield-detected spectra from the true absorption can be well modeled using the state-dependence of the fluorescence yield, and discuss implications for the application of our concept to biological samples