RESUMO
Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Cálcio/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/ultraestrutura , Membrana Celular/química , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Cristalização , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , MutagêneseRESUMO
A novel experimental setup is presented for resonant inelastic X-ray scattering investigations of solid and liquid samples in the soft X-ray region for studying the complex electronic configuration of (bio)chemical systems. The uniqueness of the apparatus is its high flexibility combined with optimal energy resolution and energy range ratio. The apparatus enables investigation of chemical analyses, which reflects the chemical imprints. The endstation is composed of a main sample chamber, a sample holder for either solid or liquid jet delivery system, and a soft X-ray grating spectrometer for 210-1250â eV with a resolving power of â¼1000. It combines for the first time liquid jet technology with a soft X-ray spectrometer based on the variable line spacing principle. This setup was commissioned at the soft X-ray beamline P04 at PETRAâ III of the Deutsches Elektronen-Synchrotron in Hamburg which is currently the most brilliant storage-ring-based X-ray radiation source in the world. The first results of liquid and solid samples show that this setup allows the detection of photons across an energy range of â¼300â eV. This covers simultaneously the emission lines of life-important elements like carbon, nitrogen and oxygen in a shot-based procedure.
RESUMO
Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.
Assuntos
Domínio Catalítico , Guanilato Ciclase/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The two major challenges in synchrotron size-exclusion chromatography coupled in-line with small-angle x-ray scattering (SEC-SAXS) experiments are the overlapping peaks in the elution profile and the fouling of radiation-damaged materials on the walls of the sample cell. In recent years, many post-experimental analyses techniques have been developed and applied to extract scattering profiles from these problematic SEC-SAXS data. Here, we present three modes of data collection at the BioSAXS Beamline 4-2 of the Stanford Synchrotron Radiation Lightsource (SSRL BL4-2). The first mode, the High-Resolution mode, enables SEC-SAXS data collection with excellent sample separation and virtually no additional peak broadening from the UHPLC UV detector to the x-ray position by taking advantage of the low system dispersion of the UHPLC. The small bed volume of the analytical SEC column minimizes sample dilution in the column and facilitates data collection at higher sample concentrations with excellent sample economy equal to or even less than that of the conventional equilibrium SAXS method. Radiation damage problems during SEC-SAXS data collection are evaded by additional cleaning of the sample cell after buffer data collection and avoidance of unnecessary exposures through the use of the x-ray shutter control options, allowing sample data collection with a clean sample cell. Therefore, accurate background subtraction can be performed at a level equivalent to the conventional equilibrium SAXS method without requiring baseline correction, thereby leading to more reliable downstream structural analysis and quicker access to new science. The two other data collection modes, the High-Throughput mode and the Co-Flow mode, add agility to the planning and execution of experiments to efficiently achieve the user's scientific objectives at the SSRL BL4-2.
Assuntos
Síncrotrons , Difração de Raios X , Espalhamento a Baixo Ângulo , Cromatografia em GelRESUMO
Although amphiphilic cyclodextrin derivatives (ACDs) serve as valuable building blocks for nanomedicine formulations, their widespread production still encounters various challenges, limiting large-scale manufacturing. This work focuses on a robust alternative pathway using mineral base catalysis to transesterify ß-cyclodextrin with long-chain vinyl esters, yielding ACD with modular and controlled hydrocarbon chain grafting. ACDs with a wide range of degrees of substitution (DS) were reliably synthesized, as indicated by extensive physicochemical characterization, including MALDI-TOF mass spectrometry. The influence of various factors, including the type of catalyst and the length of the hydrocarbon moiety of the vinyl ester, was studied in detail. ACDs were assessed for their ability to form colloidal suspensions by nanoprecipitation, with or without PEGylated phospholipid. Small-angle X-ray scattering and cryo-electron microscopy revealed the formation of nanoparticles with distinct ultrastructures depending on the DS: an onion-like structure for low and very high DS, and reversed hexagonal organization for DS between 4.5 and 6.1. We confirmed the furtivity of the PEGylated versions of the nanoparticles through complement activation experiments and that they were well tolerated in-vivo on a zebrafish larvae model after intravenous injection. Furthermore, a biodistribution experiment showed that the nanoparticles left the bloodstream within 10 h after injection and were phagocytosed by macrophages.
Assuntos
Ciclodextrinas , Nanopartículas , Animais , Ciclodextrinas/química , Microscopia Crioeletrônica/métodos , Distribuição Tecidual , Peixe-Zebra , Nanopartículas/química , Ésteres , Hidrocarbonetos , PolietilenoglicóisRESUMO
Supercritical fluids (SCFs) can be found in a variety of environmental and industrial processes. They exhibit an anomalous thermodynamic behavior, which originates from their fluctuating heterogeneous micro-structure. Characterizing the dynamics of these fluids at high temperature and high pressure with nanometer spatial and picosecond temporal resolution has been very challenging. The advent of hard x-ray free electron lasers has enabled the development of novel multi-pulse ultrafast x-ray scattering techniques, such as x-ray photon correlation spectroscopy (XPCS) and x-ray pump x-ray probe (XPXP). These techniques offer new opportunities for resolving the ultrafast microscopic behavior in SCFs at unprecedented spatiotemporal resolution, unraveling the dynamics of their micro-structure. However, harnessing these capabilities requires a bespoke high-pressure and high-temperature sample system that is optimized to maximize signal intensity and address instrument-specific challenges, such as drift in beamline components, x-ray scattering background, and multi-x-ray-beam overlap. We present a pressure cell compatible with a wide range of SCFs with built-in optical access for XPCS and XPXP and discuss critical aspects of the pressure cell design, with a particular focus on the design optimization for XPCS.
RESUMO
Disentangling human brain connectivity requires an accurate description of nerve fiber trajectories, unveiled via detailed mapping of axonal orientations. However, this is challenging because axons can cross one another on a micrometer scale. Diffusion magnetic resonance imaging (dMRI) can be used to infer axonal connectivity because it is sensitive to axonal alignment, but it has limited spatial resolution and specificity. Scattered light imaging (SLI) and small-angle X-ray scattering (SAXS) reveal axonal orientations with microscopic resolution and high specificity, respectively. Here, we apply both scattering techniques on the same samples and cross-validate them, laying the groundwork for ground-truth axonal orientation imaging and validating dMRI. We evaluate brain regions that include unidirectional and crossing fibers in human and vervet monkey brain sections. SLI and SAXS quantitatively agree regarding in-plane fiber orientations including crossings, while dMRI agrees in the majority of voxels with small discrepancies. We further use SAXS and dMRI to confirm theoretical predictions regarding SLI determination of through-plane fiber orientations. Scattered light and X-ray imaging can provide quantitative micrometer 3D fiber orientations with high resolution and specificity, facilitating detailed investigations of complex fiber architecture in the animal and human brain.
Assuntos
Encéfalo , Imagem de Difusão por Ressonância Magnética , Animais , Humanos , Chlorocebus aethiops , Raios X , Espalhamento a Baixo Ângulo , Difração de Raios X , Imagem de Difusão por Ressonância Magnética/métodos , Encéfalo/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodosRESUMO
Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals originating from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double- and triple-crossing fiber geometries, and we then apply the method in mouse, pig, vervet monkey, and human brains. We compare results to polarized light imaging (3D-PLI), tracer experiments, and to outputs from diffusion MRI that sometimes fails to detect crossings. Given its specificity, capability of 3-dimensional sampling and high resolution, SAXS could serve as a ground truth for validating fiber orientations derived using diffusion MRI as well as microscopy-based methods. STATEMENT OF SIGNIFICANCE: To study how the nerve fibers in our brain are interconnected, scientists need to visualize their trajectories, which often cross one another. Here, we show the unique capacity of small-angle X-ray scattering (SAXS) to study these fiber crossings without use of labeling, taking advantage of SAXS's specificity to myelin - the insulating sheath that is wrapped around nerve fibers. We use SAXS to detect double and triple crossing fibers and unveil intricate crossings in mouse, pig, vervet monkey, and human brains. This non-destructive method can uncover complex fiber trajectories and validate other less specific imaging methods (e.g., MRI or microscopy), towards accurate mapping of neuronal connectivity in the animal and human brain.
Assuntos
Encéfalo , Humanos , Animais , Camundongos , Suínos , Chlorocebus aethiops , Haplorrinos , Espalhamento a Baixo Ângulo , Raios X , Difração de Raios X , Encéfalo/diagnóstico por imagemRESUMO
Mussels use byssal threads to secure themselves to rocks and as shock absorbers during cyclic loading from wave motion. Byssal threads combine high strength and toughness with extensibility of nearly 200%. Researchers attribute tensile properties of byssal threads to their elaborate multi-domain collagenous protein cores. Because the elastic properties have been previously scrutinized, we instead examined byssal thread viscoelastic behaviour, which is essential for withstanding cyclic loading. By targeting protein domains in the collagenous core via chemical treatments, stress relaxation experiments provided insights on domain contributions and were coupled with in situ small-angle X-ray scattering to investigate relaxation-specific molecular reorganizations. Results show that when silk-like domains in the core were disrupted, the stress relaxation of the threads decreased by nearly 50% and lateral molecular spacing also decreased, suggesting that these domains are essential for energy dissipation and assume a compressed molecular rearrangement when disrupted. A generalized Maxwell model was developed to describe the stress relaxation response. The model predicts that maximal damping (energy dissipation) occurs at around 0.1 Hz which closely resembles the wave frequency along the California coast and implies that these materials may be well adapted to the cyclic loading of the ambient conditions.
Assuntos
Bivalves , Animais , Bivalves/química , Seda , SoftwareRESUMO
Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1â Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5â Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2â Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.
Assuntos
Benchmarking , Proteínas , Espalhamento a Baixo Ângulo , Difração de Raios X , Consenso , Reprodutibilidade dos Testes , Proteínas/química , SolventesRESUMO
Langerhans' cells (LCs) represent a specific subset of dendritic cells (DCs) which are important for detecting and processing pathogens that penetrate the skin and epithelial barriers. The aim of our study was to explain what makes their in vitro counterparts - monocyte-derived Langerhans'-like cells (MoLCs) - unique compared with monocyte-derived dendritic cells (MoDCs). Immature MoDCs were generated by incubating peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. The addition of transforming growth factor-ß (TGF-ß) to this cytokine cocktail resulted in the generation of MoLCs. MoLCs showed a lower expression of CD83, CD86, HLA-DR and CCR7 compared with MoDCs, regardless of their maturational status. Both immature and mature MoLCs secreted higher quantities of IL-23 compared with MoDCs and this finding correlated with a higher secretion of IL-17 in co-culture of MoLCs with allogeneic CD4(+) T cells. Mature MoLCs, which produced higher levels of IL-12 and lower levels of IL-10 compared with mature MoDCs, were more potent at inducing interferon-γ (IFN-γ) production by CD4(+) T cells in the co-culture system. In conclusion, the finding that mature MoLCs stimulate stronger T-helper 1 and T-helper 17 immune responses than mature MoDCs, makes them better candidates for use in the preparation of anti-tumour DC vaccines.
Assuntos
Citocinas/imunologia , Células Dendríticas/imunologia , Células de Langerhans/imunologia , Monócitos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/citologia , Humanos , Imuno-Histoquímica , Células de Langerhans/citologia , Ativação Linfocitária , Monócitos/citologiaRESUMO
Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin's nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method's sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Tomografia Computadorizada por Raios X/métodos , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Sistema Nervoso Central/diagnóstico por imagem , Pré-Escolar , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas da Mielina/metabolismo , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Neuroimagem/métodos , Estudo de Prova de Conceito , Espalhamento a Baixo Ângulo , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
Dequalinium (DQ) has been proposed as a mitochondrial targeting ligand for nanomedicines, including liposomes, given the implication of these organelles in many diseases. This original study focuses on the interactions of DQ with phosphatidylcholine bilayers during the formation of liposomes. Firstly, PEGylated liposomes suitable for drug delivery were studied and were found to be more stable when made in water than in phosphate-buffered saline, emphasizing the role of electrostatic interactions between positive charges on DQ and the polar head groups of the lipids. To gain more information, differential scanning calorimetry, small- and wide-angle X-ray scattering and diffraction, 31P and 2H NMR spectroscopy and freeze-fracture electron microscopy were performed on dimyristoylphosphatidylcholine (DMPC) model membranes in the presence of DQ. This molecule was shown to be located at the level of polar head groups and to induce electrostatic repulsions between adjacent lipid bilayers leading to membrane budding in water. These findings indicate that DQ is not completely inert towards lipid membranes and therefore is not an ideal candidate for encapsulation in liposomes. Overall, our work stresses the necessity for thorough physico-chemical characterization to better understand the mechanisms underlying the development of nanomedicines.
Assuntos
Dequalínio/química , Bicamadas Lipídicas/química , Lipídeos/química , Mitocôndrias/química , Nanomedicina , Fosfatidilcolinas/química , Estrutura MolecularRESUMO
The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.
Assuntos
Bactérias/química , Fitocromo/química , Transdução de SinaisRESUMO
In the current work, X-ray emission spectra of aqueous solutions of different inorganic salts within the Hofmeister series are presented. The results reflect the direct interaction of the ions with the water molecules and therefore, reveal general properties of the salt-water interactions. Within the experimental precision a significant effect of the ions on the water structure has been observed but no ordering according to the structure maker/structure breaker concept could be mirrored in the results indicating that the Hofmeister effect-if existent-may be caused by more complex interactions.
Assuntos
Íons/química , Modelos Químicos , Sais/química , Água/química , Ligação de Hidrogênio , Cloreto de Magnésio/química , Oxigênio/química , Cloreto de Sódio/química , Soluções , Espectrometria por Raios XRESUMO
Targeting the endosomal Toll-like receptors (TLRs) by specific agonists seems to be a promising tool for stimulation of the immunogenicity of dendritic cells (DCs). Since the functional outcome upon the engagement of TLRs may be different, the aim of our study was to examine if and how different concentrations of 7-thia-8-oxo-guanosine (7-TOG), a selective TLR7 agonist, influence differentiation, maturation and functions of human monocyte-derived DCs (MoDCs) and if its effects on MoDCs could be modulated by co-ligation of TLR3. Immature MoDCs were treated with different concentrations of 7-TOG (25, 100 and 250 µmol/L) alone, or together with polyinosinic:polycytidylic acid, Poly (I:C) (10 ng/mL), a selective TLR3 agonist, for an additional 48 h. We showed that the highest concentration of 7-TOG stimulated the differentiation, maturation and allostimulatory capability of MoDCs. These changes were accompanied by an increased production of interleukin 12 (IL-12) and induction of T helper (Th)1 and Th17 immune responses. Both Th responses were significantly augmented by additional stimulation of MoDCs with Poly (I:C). The treatment of MoDCs with the intermediate concentration of 7-TOG resulted in the up-regulation of co-stimulatory molecule (CD86) and increased production of IL-1ß and IL-6 by MoDCs, followed by the stimulation of the Th17 immune response. The lowest concentration of 7-TOG down-regulated the expression of CD40 on MoDCs and potentiated the Th2 immune response. The Th2 response was not significantly modulated by additional treatment of MoDCs with Poly (I:C), but this combination of TLR3/TLR7 agonists also stimulated both Th1 and Th17 responses. In conclusion, our results show that 7-TOG influences the phenotype and functions of MoDCs in a dose-dependent manner and suggests that fine-tuned signaling through TLR7 may be modified by the engagement of TLR3, resulting in a different outcome of immune response.
Assuntos
Células Dendríticas/efeitos dos fármacos , Guanosina/análogos & derivados , Poli I-C/farmacologia , Células Cultivadas , Células Dendríticas/citologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Guanosina/farmacologia , Humanos , Interleucinas/metabolismo , Teste de Cultura Mista de Linfócitos , Receptores Toll-Like/agonistas , Receptores Toll-Like/fisiologiaRESUMO
Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.
Assuntos
Células Dendríticas/imunologia , Lectinas Tipo C/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptores Toll-Like/metabolismo , Antígenos CD/metabolismo , Humanos , Imunoterapia/métodos , Células de Langerhans/imunologia , Lectinas de Ligação a Manose/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Neoplasias/terapia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
We present a flexible and compact experimental setup that combines an in vacuum liquid jet with an x-ray emission spectrometer to enable static and femtosecond time-resolved resonant inelastic soft x-ray scattering (RIXS) measurements from liquids at free electron laser (FEL) light sources. We demonstrate the feasibility of this type of experiments with the measurements performed at the Linac Coherent Light Source FEL facility. At the FEL we observed changes in the RIXS spectra at high peak fluences which currently sets a limit to maximum attainable count rate at FELs. The setup presented here opens up new possibilities to study the structure and dynamics in liquids.