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A GCxGC-MS system was employed with a non-polar × mid-polar column set for the metabolic non-target analysis of Cobetia marina, the model bacteria for marine biofouling. C. marina was treated with ozone to investigate the intracellular metabolic state change under oxidative stress. A minimal inhibitory concentration test was involved to guarantee that the applied ozone dosages were not lethal for the cells. In this study, non-target analyses were performed to identify the metabolites according to the NIST database. As a result, over 170 signals were detected under normal living conditions including 35 potential metabolites. By the comparison of ozone-treated and non-treated samples, five compounds were selected to describe observed trends of signals in the contour plots. Oleic acid exhibited a slight growth by increasing ozone dosage. In contrast, other metabolites such as the amino acid l-proline showed less abundance after ozone treatment, which was more evident once ozone dosage was raised. Thus, this work could provide a hint for searching for up/downregulating factors in such environmental stress conditions for C. marina.

Frontiers in pharmacology 12, 769739 (2021). doi:10.3389/fphar.2021.769739, Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a “dirty drug” model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development., Published by Frontiers Media, Lausanne

ChemSystemsChem 3(3), e2000050 (2021). doi:10.1002/syst.202000050, Huntington's disease (HD) is one of nine neurodegenerative disorders associated with an extension of polyglutamine (polyQ) in proteins. In HD, the polyQ tract in the huntingtin protein is extended beyond a threshold of 38 amino acids leading to the formation of amyloidal structures in the cytoplasm and nucleus. We investigated here the structure of Htt (Huntingtin) amyloid fibrils in cellulo with nanoprobe small angle X-ray scattering. As these measurements were performed under cryogenic conditions, the information is obtained on the aggregates in their natural, hydrated environment without the need of staining and chemical fixation. We also could show the presence of oligomer structures not visible in fluorescence microscopy. Structural information on repetitive units inside of Htt inclusion bodies was determined from the SAXS data and compared to electron microscopy images. The results suggest that nanoprobe cryo-SAXS can serve as powerful tool to investigate the kinetics of amyloid aggregate formation inside cells and to understand how fibril formation can be influenced by drugs and other external stimuli., Published by Wiley-VCH Verlag, Weinheim

Biointerphases 16(1), 011004 (1-8) (2021). doi:10.1116/6.0000593, X-ray fluorescence analysis enables the study of trace element distributions in biological specimens. When this analysis is done under cryogenic conditions, cells are cryofixed as closely as possible to their natural physiological state, and the corresponding intracellular elemental densities can be analyzed. Details about the experimental setup used for analysis at the P06 beamline at Petra III, DESY and the used cryo-transfer system are described in this work. The system was applied to analyze the elemental distribution in single HeLa cells, a cell line frequently used in a wide range of biological applications. Cells adhered to silicon nitride substrates were cryoprotected within an amorphous ice matrix. Using a continuous scanning scheme and a KB x-ray focus, the distribution of elements in the cells was studied. We were able to image the intracellular potassium and zinc levels in HeLa cells as two key elements relevant for the physiology of cells., Published by AVS, New York, NY

Journal of synchrotron radiation 27(3), 625 - 632 (2020). doi:10.1107/S1600577520002192, The unique diagnostic possibilities of X-ray diffraction, small X-ray scattering and phase-contrast imaging techniques applied with high-intensity coherent X-ray synchrotron and X-ray free-electron laser radiation can only be fully realized if a sufficient dynamic range and/or spatial resolution of the detector is available. In this work, it is demonstrated that the use of lithium fluoride (LiF) as a photoluminescence (PL) imaging detector allows measuring of an X-ray diffraction image with a dynamic range of ∼107 within the sub-micrometre spatial resolution. At the PETRA III facility, the diffraction pattern created behind a circular aperture with a diameter of 5 µm irradiated by a beam with a photon energy of 500 eV was recorded on a LiF crystal. In the diffraction pattern, the accumulated dose was varied from 1.7 × 10$^5$ J cm$^{−3}$ in the central maximum to 2 × 10$^{−2}$ J cm$^{−3}$ in the 16th maximum of diffraction fringes. The period of the last fringe was measured with 0.8 µm width. The PL response of the LiF crystal being used as a detector on the irradiation dose of 500 eV photons was evaluated. For the particular model of laser-scanning confocal microscope Carl Zeiss LSM700, used for the readout of the PL signal, the calibration dependencies on the intensity of photopumping (excitation) radiation (λ = 488 nm) and the gain have been obtained., Published by Wiley-Blackwell, [S.l.]

Nanoporous (np) gold is a promising catalyst material for selective oxidation reactions. Especially the addition of oxide deposits like ceria (CeO2) promises enhanced morphological stability for high temperature applications. Describing such temperature induced morphological changes in porous materials is challenging. Here, X-ray nanoanalysis is particularly promising due to the high penetration depth that allows studying of the bulk properties with high spatial sensitivity. We applied soft X-ray small angle scattering (SAXS) to determine temperature induced structural changes in nanoporous gold catalysts. The results show that CeO2 deposits enhance the temperature stability of the nanoporous gold catalyst. Moreover, we demonstrate the ability of soft X-rays to selectively provide structural information on the stabilizing cerium oxide deposits via resonant, anomalous SAXS (ASAXS) measurements at the cerium M-edge, revealing no growth of the ceria particles.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was investigated in different organic cosolvents by means of kinetic and calorimetric measurements, molecular dynamics simulations, and small-angle X-ray scattering. The combined experimental and theoretical techniques were essential to complement each other's limitations in the investigation of the complex interaction pattern between the enzyme, different solvent types, and concentrations. In this way, the underlying mechanisms for the loss of enzyme activity in different water-miscible solvents could be elucidated. These include direct inhibitory effects onto the active center and structural distortions.