Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

Multiple-core-hole resonance spectroscopy with ultraintense X-ray pulses.

Understanding the interaction of intense, femtosecond X-ray pulses with heavy atoms is crucial for gaining insights into the structure and dynamics of matter. One key aspect of nonlinear light-matter interaction was, so far, not studied systematically at free-electron lasers-its dependence on the photon energy. Here, we use resonant ion spectroscopy to map out the transient electronic structures occurring during the complex charge-up pathways of xenon. Massively hollow atoms featuring up to six simultaneous core holes determine the spectra at specific photon energies and charge states. We also illustrate how different X-ray pulse parameters, which are usually intertwined, can be partially disentangled. The extraction of resonance spectra is facilitated by the possibility of working with a constant number of photons per X-ray pulse at all photon energies and the fact that the ion yields become independent of the peak fluence beyond a saturation point. Our study lays the groundwork for spectroscopic investigations of transient atomic species in exotic, multiple-core-hole states that have not been explored previously.

XFEL Microcrystallography of Self-Assembling Silver n-Alkanethiolates.

New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd-even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd-even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd-even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.

On Experiential Loneliness.

Presumably, everyone has, at some point in their lives, felt lonely. Loneliness is, in that particular sense, omnipresent. What it feels like to be lonely can, however, vary significantly. Loneliness is far from being a homogeneous phenomenon. Different kinds of loneliness need to be distinguished, considering its causes, contexts, a person's capacities to cope with it, and many other factors. This paper introduces the notion of a specific kind of loneliness: experiential loneliness. Experiential loneliness, it will be argued, consists in particular ways of experiencing the world, oneself, and others. Although feelings of being lonely in one way or another can emanate from one's experience of the world being structured in a particular manner, such kinds of loneliness need not-at least, not always and the whole time-lead to emotional feelings that are concerned with one's loneliness or the lack of meaningful social relationship. Loneliness can give rise to quite different emotional feelings that sometimes even cover up their provenience from underlying experiential loneliness. The notion of experiential loneliness, it is suggested, helps to tie back certain styles of thinking, desires, feelings, and behaviors to contexts of loneliness. Moreover, it will be argued that the notion can also elucidate the development of feelings of being lonely in contexts in which others are not only around but also available. To develop and enrich the notion of experiential loneliness as well as to exemplify its usefulness, a closer look will be taken at the case of borderline personality disorder, a condition in which sufferers are often plagued by loneliness.

Harmonic radiation contribution and X-ray transmission at the Small Quantum Systems instrument of European XFEL.

Transmission measurements of the soft X-ray beamline to the Small Quantum Systems (SQS) scientific instrument at the SASE3 undulator of European XFEL are presented. Measurements are reported for a wide range of photon energies (650 eV to 2400 eV), using X-ray gas monitors as well as a bolometric radiometer. The results are in good agreement with simulations for the beam transport and show a transmission of up to 80% over the whole photon energy range. The contribution of second- and third-harmonic radiation of the soft X-ray undulator is determined at selected photon energies by performing transmission measurements using a gas absorber to provide variable attenuation of the incoming photon flux. A comparison of the results with semi-analytic calculations for the generation of free-electron laser pulses in the SASE3 undulator reveals an influence of apertures along the beam transport on the exact harmonic content to be accounted for at the experiment. The second-harmonic content is measured to be in the range of 0.1% to 0.3%, while the third-harmonic contributed a few percent to the SASE3 emission. For experiments at the SQS instrument, these numbers can be reduced through specific selections of the mirror reflection angles.

The beam transport system for the Small Quantum Systems instrument at the European XFEL: optical layout and first commissioning results.

The Small Quantum Systems instrument is one of the six operating instruments of the European XFEL, dedicated to the atomic, molecular and cluster physics communities. The instrument started its user operation at the end of 2018 after a commissioning phase. The design and characterization of the beam transport system are described here. The X-ray optical components of the beamline are detailed, and the beamline performances, transmission and focusing capabilities are reported. It is shown that the X-ray beam can be effectively focused as predicted by ray-tracing simulations. The impact of non-ideal X-ray source conditions on the focusing performances is discussed.

Recall bias of students' affective experiences in adolescence: The role of personality and internalizing behavior.

INTRODUCTION: Adolescence is characterized by multiple biopsychosocial changes, associated with a high intraindividual variability of emotional experiences. Previous findings suggest that this intraindividual variability is reflected in a recall bias of adolescents' emotion reports. However, corresponding findings are scarce and inconclusive. Studies on predictors of recall bias in adulthood indicate that personality traits, especially neuroticism and extraversion, as well as specific internalizing disorders might affect recall bias of emotion reports. METHODS: The sample consists of 118 Swiss adolescent students in grade 8 and 9 (Mage = 15.15, SDage = 0.89). The students' momentary affective experience was recorded using smartphones over seven consecutive days in situ at 42 randomly generated occasions (six per day), with a total of 1059 protocols on current events. At the end of the experience-sampling phase, students filled out an online questionnaire, providing information about their personality and typical behavior as well as their retrospective affective experience. In addition, the students' behavior was evaluated by their teachers. We applied two-level structural equation modeling with latent difference variables. RESULTS: Adolescents high in extraversion showed retrospective overestimation of positive affective experiences and underestimation of negative affective experiences. Adolescents with high neuroticism tended to overestimate negative affect retrospectively, showing no significant effects for positive affect. However, internalizing behavior did not predict a negative recall bias in adolescents' affective experience. CONCLUSIONS: Retrospective self-reports about adolescents' affective experience are biased by relatively stable individual factors, whereas less stable individual factors did not seem to have any influence.

Separation and identification of a complex flurbiprofen-polyethylene glycol mono- and diester mixture via a hyphenated HPLC-DAD-HRMS/SPE NMR system.

We report the use of a hyphenated HPLC-DAD(diode array detector)-HRMS/SPE NMR system for the separation and isolation of a complex mixture of esters, containing substances with very similar LC retention times. The literature known mono- and diesters of the drug Flurbiprofen and polyethylene glycol, which form a large number of substances with varying chain lengths, were chosen for this study. We demonstrate the use of this hyphenated system to quickly and effectively isolate sixteen of these highly similar individual esters in an automated fashion, demonstrating its applicability in standard pharmaceutical analysis and quality control of drugs. Both, synthetic solutions of these esters and extracts from Flurbiprofen lozenges were used for this purpose. By the sole use of this system, the individual compounds were isolated and UV, HRMS and 1D and 2D NMR data could be collected, enabling the identification and differentiation of the individual esters.

Electronic quantum coherence in glycine molecules probed with ultrashort x-ray pulses in real time.

Here, we use x-rays to create and probe quantum coherence in the photoionized amino acid glycine. The outgoing photoelectron leaves behind the cation in a coherent superposition of quantum mechanical eigenstates. Delayed x-ray pulses track the induced coherence through resonant x-ray absorption that induces Auger decay and by photoelectron emission from sequential double photoionization. Sinusoidal temporal modulation of the detected signal at early times (0 to 25 fs) is observed in both measurements. Advanced ab initio many-electron simulations allow us to explain the first 25 fs of the detected coherent quantum evolution in terms of the electronic coherence. In the kinematically complete x-ray absorption measurement, we monitor its dynamics for a period of 175 fs and observe an evolving modulation that may implicate the coupling of electronic to vibronic coherence at longer time scales. Our experiment provides a direct support for the existence of long-lived electronic coherence in photoionized biomolecules.

Affective Instability and Emotion Dysregulation as a Social Impairment.

Borderline personality disorder is a complex psychopathological phenomenon. It is usually thought to consist in a vast instability of different aspects that are central to our experience of the world, and to manifest as "a pervasive pattern of instability of interpersonal relationships, self-image, and affects, and marked impulsivity" [American Psychiatric Association (APA), 2013, p. 663]. Typically, of the instability triad-instability in (1) self, (2) affect and emotion, and (3) interpersonal relationships-only the first two are described, examined, and conceptualized from an experiential point of view. In this context, disorders of self have often motivated analyses of self-experience and the sense of self, affective disorders have been frequently considered in the light of emotional experience and its phenomenological structure. Patterns in the phenomenology of social experience have found comparatively little traction when it comes to the conceptualization of the interpersonal disturbances in borderline. In this paper, I argue that interpersonal instability in borderline consists in much more than fragile and shifting relationships but, most importantly, also involves certain styles in experiencing others. These styles, I suggest, may play an explanatory role for the borderline-typical patterns of interpersonal turmoil and so deserve more attention. To better describe and understand these styles, I explore the phenomenological structure of borderline affective instability and discuss the implications it might have for how a person experiences and relates to other people. Considering core aspects of borderline affective instability, such as alexithymia, emotional contagion, emotion dysregulation, and chronic emptiness, I propose borderline can be interpreted as a disturbance of interaffective exchange, which gives rise to certain ways of experiencing others that imply a social impairment.

High-resolution electron time-of-flight spectrometers for angle-resolved measurements at the SQS Instrument at the European XFEL.

A set of electron time-of-flight spectrometers for high-resolution angle-resolved spectroscopy was developed for the Small Quantum Systems (SQS) instrument at the SASE3 soft X-ray branch of the European XFEL. The resolving power of this spectrometer design is demonstrated to exceed 10 000 (E/ΔE), using the well known Ne 1s-13p resonant Auger spectrum measured at a photon energy of 867.11 eV at a third-generation synchrotron radiation source. At the European XFEL, a width of ∼0.5 eV full width at half-maximum for a kinetic energy of 800 eV was demonstrated. It is expected that this linewidth can be reached over a broad range of kinetic energies. An array of these spectrometers, with different angular orientations, is tailored for the Atomic-like Quantum Systems endstation for high-resolution angle-resolved spectroscopy of gaseous samples.

Borderline We-Space? The Phenomenology of the Background of Safety in Borderline Personality Disorder.

Borderline personality disorder (BPD) is a severe psychiatric condition characterized by instability in identity, relationships, and affect. Individuals, with BPD typically lack a coherent sense of self, are highly sensitive to interpersonal stressors, experience intense fluctuations in mood, and frequently engage in impulsive and self-destructive behaviors. Although both empirical research and development of effective psychotherapy have evidently progressed over the past years, many aspects regarding the structure of experience and the life-world typical for persons with BPD are not yet fully understood. Somewhat surprisingly, phenomenological psychopathology has only recently started to pay more attention to the disorder. A comprehensive elaboration of the phenomenology of BPD is therefore still lacking. This article aimed to contribute to such a phenomenological understanding by focusing on what we think is an essential aspect that has yet not been sufficiently addressed: the background of safety. To clarify what this means, we depart from Sandler's [Int J Psychoan. 1960;41:352-6] psychoanalytic concept and elaborate on it phenomenologically. This leads us to argue that the development of a background of safety requires a particular embodied presence of others, which, in turn, contributes to the constitution of a safe we-space, a shared and familiar environment providing a matrix for the experience of a stable world. However, even when established, the background of safety remains in need of a continuous reconfirmation through corresponding experiences within a sufficiently reliable and controllable environment. The background of safety is vulnerable and open to (interpersonal) disruptions like trauma or neglect. In BPD, we suggest 3 aspects regarding the phenomenology of the background of the safety need to be considered: first, typically, patients with BPD did not develop a robust background of safety in infancy; second, weakening of the background of safety gives rise to symptoms and dynamics typical for BPD; third, these symptoms and dynamics further undermine the possible development of a background of safety in adult life and thus gravitate toward a petrification of the borderline condition, a "stable instability." To conclude, we examine whether this concept should be understood as a trouble générateur and, last, consider its clinical implications.

Isolation, Identification and Structural Verification of a Methylene-Bridged Naloxone "Dimer" Formed by Formaldehyde.

We report the isolation and characterization of a methylene bridged "dimer" of the opioid antagonist Naloxone, previously detected in experimental Buprenorphine-Naloxone oral films. This compound was found to form via an aldol addition followed by a condensation reaction under acidic conditions between two units of Naloxone and one unit of formaldehyde. HPLC-UV-HRMS analysis revealed the formation of three individual stereoisomers during this reaction, which were separately isolated using solid-phase extraction. These isomers were shown to freely react into one another in solvent, forming an equilibrium. The structure of the unknown compound was determined via HRMS spectrometry and 1D and 2D NMR spectroscopy.

A localized view on molecular dissociation via electron-ion partial covariance.

Inner-shell photoelectron spectroscopy provides an element-specific probe of molecular structure, as core-electron binding energies are sensitive to the chemical environment. Short-wavelength femtosecond light sources, such as Free-Electron Lasers (FELs), even enable time-resolved site-specific investigations of molecular photochemistry. Here, we study the ultraviolet photodissociation of the prototypical chiral molecule 1-iodo-2-methylbutane, probed by extreme-ultraviolet (XUV) pulses from the Free-electron LASer in Hamburg (FLASH) through the ultrafast evolution of the iodine 4d binding energy. Methodologically, we employ electron-ion partial covariance imaging as a technique to isolate otherwise elusive features in a two-dimensional photoelectron spectrum arising from different photofragmentation pathways. The experimental and theoretical results for the time-resolved electron spectra of the 4d3/2 and 4d5/2 atomic and molecular levels that are disentangled by this method provide a key step towards studying structural and chemical changes from a specific spectator site.

Resonance-Enhanced Multiphoton Ionization in the X-Ray Regime.

Here, we report on the nonlinear ionization of argon atoms in the short wavelength regime using ultraintense x rays from the European XFEL. After sequential multiphoton ionization, high charge states are obtained. For photon energies that are insufficient to directly ionize a 1s electron, a different mechanism is required to obtain ionization to Ar^{17+}. We propose this occurs through a two-color process where the second harmonic of the FEL pulse resonantly excites the system via a 1s→2p transition followed by ionization by the fundamental FEL pulse, which is a type of x-ray resonance-enhanced multiphoton ionization (REMPI). This resonant phenomenon occurs not only for Ar^{16+}, but also through lower charge states, where multiple ionization competes with decay lifetimes, making x-ray REMPI distinctive from conventional REMPI. With the aid of state-of-the-art theoretical calculations, we explain the effects of x-ray REMPI on the relevant ion yields and spectral profile.

Timing and X-ray pulse characterization at the Small Quantum Systems instrument of the European X-ray Free Electron Laser.

This contribution presents the initial characterization of the pump-probe performance at the Small Quantum Systems (SQS) instrument of the European X-ray Free Electron Laser. It is demonstrated that time-resolved experiments can be performed by measuring the X-ray/optical cross-correlation exploiting the laser-assisted Auger decay in neon. Applying time-of-arrival corrections based on simultaneous spectral encoding measurements allow us to significantly improve the temporal resolution of this experiment. These results pave the way for ultrafast pump-probe investigations of gaseous media at the SQS instrument combining intense and tunable soft X-rays with versatile optical laser capabilities.

Development of the Commercial Manufacturing Process for Ipatasertib.

Ipatasertib is a potent small molecule Akt kinase inhibitor currently being tested in Phase III clinical trials for the treatment of metastatic castration-resistant prostate cancer and triple negative metastatic breast cancer. In this paper an overview of the development achievements towards the commercial manufacturing process is given. The convergent synthesis consists of ten steps with eight isolated intermediates and utilizes a wide range of chemical techniques and technologies to build-up this complex drug. All three stereocenters are introduced using enzyme or metal catalysis.

Suppression of X-ray-Induced Radiation Damage to Biomolecules in Aqueous Environments by Immediate Intermolecular Decay of Inner-Shell Vacancies.

The predominant reason for the damaging power of high-energy radiation is multiple ionization of a molecule, either direct or via the decay of highly excited intermediates, as, e.g., in the case of X-ray irradiation. Consequently, the molecule is irreparably damaged by the subsequent fragmentation in a Coulomb explosion. In an aqueous environment, however, it has been observed that irradiated molecules may be saved from fragmentation presumably by charge and energy dissipation mechanisms. Here, we show that the protective effect of the environment sets in even earlier than hitherto expected, namely immediately after single inner-shell ionization. By combining coincidence measurements of the fragmentation of X-ray-irradiated microsolvated pyrimidine molecules with theoretical calculations, we identify direct intermolecular electronic decay as the protective mechanism, outrunning the usually dominant Auger decay. Our results demonstrate that such processes play a key role in charge delocalization and have to be considered in investigations and models on high-energy radiation damage in realistic environments.

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids.

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2-5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12-90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.

Site-specific interrogation of an ionic chiral fragment during photolysis using an X-ray free-electron laser.

Short-wavelength free-electron lasers with their ultrashort pulses at high intensities have originated new approaches for tracking molecular dynamics from the vista of specific sites. X-ray pump X-ray probe schemes even allow to address individual atomic constituents with a 'trigger'-event that preludes the subsequent molecular dynamics while being able to selectively probe the evolving structure with a time-delayed second X-ray pulse. Here, we use a linearly polarized X-ray photon to trigger the photolysis of a prototypical chiral molecule, namely trifluoromethyloxirane (C3H3F3O), at the fluorine K-edge at around 700 eV. The created fluorine-containing fragments are then probed by a second, circularly polarized X-ray pulse of higher photon energy in order to investigate the chemically shifted inner-shell electrons of the ionic mother-fragment for their stereochemical sensitivity. We experimentally demonstrate and theoretically support how two-color X-ray pump X-ray probe experiments with polarization control enable XFELs as tools for chiral recognition.