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Imaging the charge flow in photoexcited molecules would provide key information on photophysical and photochemical processes. Here the authors demonstrate tracking in real time after photoexcitation the change in charge density at a specific site of 2-thiouracil using time-resolved X-ray photoelectron spectroscopy.

The method of time-resolved XUV photoelectron spectroscopy is applied in a pump–probe experiment on a liquid micro-jet. We investigate how the XUV energy spectra of photoelectrons are influenced by the space charge created due to ionization of the liquid medium by the pump laser pulse. XUV light from high-order harmonic generation is used to probe the electron population of the valence shell of iron hexacyanide in water. By exposing the sample to a short UV pump pulse of 266 nm wavelength and ∼55 fs duration, we observe an energy shift of the spectral component associated with XUV ionization from the Fe 3d(t _2 _g ) orbital as well as a shift of the water spectrum. Depending on the sequence of the pump and probe pulses, the arising energy shift of photoelectrons acquires a positive or negative value. It exhibits a sharp positive peak at small time delays, which facilitates to determine the temporal overlap between pump and probe pulses. The negative spectral shift is due to positive charge accumulated in the liquid medium during ionization. Its dissipation is found to occur on a (sub)nanosecond time scale and has a biexponential character. A simple mean-field model is provided to interpret the observations. A comparison between the intensity dependencies of the spectral shift and the UV ionization yield shows that the space-charge effect can be significantly reduced when the pump intensity is attenuated below the saturation level of water ionization. For the given experimental conditions, the saturation intensity lies at $6\times {10}^{10}$ W cm ^−2 .

We report on strong-field ionization of dense water gas in a short infrared laser pulse. By employing a unique combination of photoelectron spectroscopy with a liquid micro-jet technique, we observe how the character of electron emission at high kinetic energies changes with the increase of the medium density. This change is associated with the process of laser-assisted electron scattering (LAES) on neighboring particles, which becomes a dominant mechanism of hot electron emission at higher medium densities. The manifestation of this mechanism is found to require densities that are orders of magnitude lower than those considered for heating the laser-generated plasmas via the LAES process. The experimental results are supported by simulations of the LAES yield with the use of the Kroll–Watson theory.

The conversion of photon energy into other energetic forms in molecules is accompanied by charge moving on ultrafast timescales. We directly observe the charge motion at a specific site in an electronically excited molecule using time-resolved x-ray photoelectron spectroscopy (TR-XPS). We extend the concept of static chemical shift from conventional XPS by the excited-state chemical shift (ESCS), which is connected to the charge in the framework of a potential model. This allows us to invert TR-XPS spectra to the dynamic charge at a specific atom. We demonstrate the power of TR-XPS by using sulphur 2p-core-electron-emission probing to study the UV-excited dynamics of 2-thiouracil. The method allows us to discover that a major part of the population relaxes to the molecular ground state within 220-250 fs. In addition, a 250-fs oscillation, visible in the kinetic energy of the TR-XPS, reveals a coherent exchange of population among electronic states., (© 2022. The Author(s).)

In this paper, we report X-ray absorption and core-level electron spectra of the nucleobase derivative 2-thiouracil at the sulfur L 1 - and L 2,3 -edges. We used soft X-rays from the free-electron laser FLASH2 for the excitation of isolated molecules and dispersed the outgoing electrons with a magnetic bottle spectrometer. We identified photoelectrons from the 2p core orbital, accompanied by an electron correlation satellite, as well as resonant and non-resonant Coster-Kronig and Auger-Meitner emission at the L 1 - and L 2,3 -edges, respectively. We used the electron yield to construct X-ray absorption spectra at the two edges. The experimental data obtained are put in the context of the literature currently available on sulfur core-level and 2-thiouracil spectroscopy.

The ultrafast photo-induced ring opening of the oxirane derivative trans-stilbene oxide has been studied through the use of ultrafast UV/UV pump-probe spectroscopy by using photo-ion detection. Single- and multiphoton probe paths and final states were identified through comparisons between UV power studies and synchrotron-based vacuum ultraviolet (VUV) single-photon ionization studies. Three major time-dependent features of the parent ion (sub-450 fs decay, (1.5±0.2) ps, and >100 ps) were observed. These decays are discussed in conjunction with the primary ring-opening mechanism of stilbene oxide, which occurs through C-C dissociation in the oxirane ring. The appearance of fragments relating to the masses of dehydrogenated diphenylmethane (167 amu) and dehydrogenated methylbenzene (90 amu) were also investigated. The appearance of the 167 amu fragment could suggest an alternative ultrafast ring-opening pathway via the dissociation of one of the C-O bonds within the oxirane ring., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12-Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation. Abbreviations : FCCS: fluorescence cross correlation spectroscopy; NVJ: nucleus-vacuole junction; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol- 3-phosphate; VICS: vacuole isolation membrane contact site.

The Atg12-Atg5/Atg16L1 complex is recruited by WIPI2b to the site of autophagosome formation. Atg16L1 is an effector of the Golgi resident GTPase Rab33B. Here we identified a minimal stable complex of murine Rab33B(30-202) Q92L and Atg16L1(153-210). Atg16L1(153-210) comprises the C-terminal part of the Atg16L1 coiled-coil domain. We have determined the crystal structure of the Rab33B Q92L/Atg16L1(153-210) effector complex at 3.47 Å resolution. This structure reveals that two Rab33B molecules bind to the diverging α-helices of the dimeric Atg16L1 coiled-coil domain. We mutated Atg16L1 and Rab33B interface residues and found that they disrupt complex formation in pull-down assays and cellular co-localization studies. The Rab33B binding site of Atg16L1 comprises 20 residues and immediately precedes the WIPI2b binding site. Rab33B mutations that abolish Atg16L binding also abrogate Rab33B association with the Golgi stacks. Atg16L1 mutants that are defective in Rab33B binding still co-localize with WIPI2b in vivo. The close proximity of the Rab33B and WIPI2b binding sites might facilitate the recruitment of Rab33B containing vesicles to provide a source of lipids during autophagosome biogenesis.

The application of site directed nucleases (SDN) for Genome Editing (GE) in plant breeding and research increases exponentially in the last few years. The main research so far was on 'proof of concept' studies or improvement of the precision and delivery of the SDN. Nevertheless, a reasonable amount of research is present on market-oriented applications for cash crops such as rice but also for commercially lesser interesting crops and vegetables. Reported field trials involving GE plants are scarce around the world and almost not existing in Europe. This is due to the regulatory landscape for GE plants, which is quite distinct and especially in the European Union very demanding. By far the most field trials involve GE rice varieties in the Asian area, followed up by tomato and other vegetables and crops., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe the further development of the RNA-guided endonuclease-in situ labeling (RGEN-ISL) method CRISPR-FISH. Using soybean and mouse chromosomes, we demonstrate that the treatment of conventionally fixed chromosomes (in ethanol or methanol:acetic acid) with 40 mM Tris-HCl (pH 9.0) for 30 minutes at 37°C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, and Nicotiana benthamiana were used to confirm the applicability of the identified CRISPR-FISH conditions also in other species., (© 2020 S. Karger AG, Basel.)

Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Genome Editing using engineered endonuclease (GEEN) systems rapidly took over the field of plant science and plant breeding. So far, Genome Editing techniques have been applied in more than fifty different plants; including model species like Arabidopsis; main crops like rice, maize or wheat as well as economically less important crops like strawberry, peanut and cucumber. These techniques have been used for basic research as proof-of-concept or to investigate gene functions in most of its applications. However, several market-oriented traits have been addressed including enhanced agronomic characteristics, improved food and feed quality, increased tolerance to abiotic and biotic stress and herbicide tolerance. These technologies are evolving at a tearing pace and especially the field of CRISPR based Genome Editing is advancing incredibly fast. CRISPR-Systems derived from a multitude of bacterial species are being used for targeted Gene Editing and many modifications have already been applied to the existing CRISPR-Systems such as (i) alter their protospacer adjacent motif (ii) increase their specificity (iii) alter their ability to cut DNA and (iv) fuse them with additional proteins. Besides, the classical transformation system using Agrobacteria tumefaciens or Rhizobium rhizogenes , other transformation technologies have become available and additional methods are on its way to the plant sector. Some of them are utilizing solely proteins or protein-RNA complexes for transformation, making it possible to alter the genome without the use of recombinant DNA. Due to this, it is impossible that foreign DNA is being incorporated into the host genome. In this review we will present the recent developments and techniques in the field of DNA-free Genome Editing, its advantages and pitfalls and give a perspective on technologies which might be available in the future for targeted Genome Editing in plants. Furthermore, we will discuss these techniques in the light of existing- and potential future regulations.

Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We report on a newly built laser-based tabletop setup which enables generation of femtosecond light pulses in the XUV range employing the process of high-order harmonic generation (HHG) in a gas medium. The spatial, spectral, and temporal characteristics of the XUV beam are presented. Monochromatization of XUV light with minimum temporal pulse distortion is the central issue of this work. Off-center reflection zone plates are shown to be advantageous when selection of a desired harmonic is carried out with the use of a single optical element. A cross correlation technique was applied to characterize the performance of the zone plates in the time domain. By using laser pulses of 25 fs length to pump the HHG process, a pulse duration of 45 fs for monochromatized harmonics was achieved in the present setup.

A newly constructed time-of-flight electron spectrometer of the magnetic bottle type is characterized for electron detection in a broad range of kinetic energies. The instrument is designed to measure the energy spectra of electrons generated from liquids excited by strong laser fields and photons in the range of extreme ultra violet and soft X-rays. Argon inner shell electrons were recorded to calibrate the spectrometer and investigate its characteristics, such as energy resolution and collection efficiency. Its energy resolution ΔE/E of 1.6% allows resolving the Ar 2p spin orbit structure at kinetic energies higher than 100 eV. The collection efficiency is determined and compared to that of the spectrometer in its field-free configuration.