Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

18 pags, 11 figs, 5 tabsHere, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.This work was supported by the National Science Foundation Science and Technology Center 'BioXFEL' through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DESC0002164 (AO, algorithm design and development) and by the National Science Foundation under contract Nos. 1551489 (AO, underlying analytical models) and DBI-2029533 (AO, functional conformations). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 to JLO. The work was also supported by funds from the National Institutes of Health grant R01 GM117342-0404. Funding and support are also acknowledged from the National Institutes of Health grant R01 GM095583, from the Biodesign Center for Applied Structural Discovery at ASU, from National Science Foundation award No. 1565180 and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. KAZ was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). This work was also supported by the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. CFEL is supported by the Gottfried Wilhelm Leibniz Program of the DFG, the 'X-probe' project funded by the European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement 637295, the European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)', ERC-2013-SyG 609920, and the Human Frontiers Science Program grant RGP0010 2017. This work is also supported by the AXSIS project funded by the European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 609920.Peer reviewe

Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses

Data for "A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS"

The application of small-angle X-ray scattering (SAXS) for high-throughput characterization of biological macromolecules in solution is limited by radiation damage. By cryocooling samples, radiation damage and required sample volumes can be reduced by orders of magnitude. However, the challenges of reproducibly creating the identically sized vitrified samples necessary for conventional background subtraction limit the widespread adoption of this method. Fixed path length silicon sample holders for cryoSAXS have been micro fabricated to address these challenges. They have low background scattering and X-ray absorption, require only 640 nl of sample, and allow reproducible sample cooling. Data collected in the sample holders from a nominal illuminated sample volume of 2.5 nl are reproducible down to q ’ 0.02 Å-1, agree with previous cryoSAXS work and are of sufficient quality for reconstructions that match measured crystal structures. These sample holders thus allow faster, more routine cryoSAXS data collection. Additional development is required to reduce sample fracturing and improve data quality at low q

Usability Testing of the Kidney Score Platform to Enhance Communication About Kidney Disease in Primary Care Settings: Qualitative Think-Aloud Study

BackgroundPatient awareness of chronic kidney disease (CKD) is low in part due to suboptimal testing for CKD among those at risk and lack of discussions about kidney disease between patients and clinicians. To bridge these gaps, the National Kidney Foundation developed the Kidney Score Platform, which is a web-based series of tools that includes resources for health care professionals as well as an interactive, dynamic patient-facing component that includes a brief questionnaire about risk factors for kidney disease, individualized assessment of risk for developing CKD, and self-management tools to manage one’s kidney disease. ObjectiveThe aim of this study is to perform usability testing of the patient component of the Kidney Score platform among veterans with and at risk for kidney disease and among clinicians working as primary care providers in Veterans Affairs administration. MethodsThink-aloud exercises were conducted, during which participants (veterans and clinicians) engaged with the platform while verbalizing their thoughts and making their perceptions, reasonings, and decision points explicit. A usability facilitator observed participants’ behaviors and probed selectively to clarify their comprehension of the tool’s instructions, content, and overall functionality. Thematic analysis on the audio-recording transcripts was performed, focusing on positive attributes, negative comments, and areas that required facilitator involvement. ResultsVeterans (N=18) were 78% (14/18) male with a mean age of 58.1 years. Two-thirds (12/18) were of non-White race/ethnicity, 28% (5/18) had laboratory evidence of CKD without a formal diagnosis, and 50% (9/18) carried a diagnosis of hypertension or diabetes. Clinicians (N=19) were 29% (5/17) male, 30% (5/17) of non-White race/ethnicity, and had a mean of 17 (range 4-32) years of experience. Veterans and clinicians easily navigated the online tool and appreciated the personalized results page as well as the inclusion of infographics to deliver key educational messages. Three major themes related to content and communication about risk for CKD emerged from the think-aloud exercises: (1) tension between lay and medical terminology when discussing kidney disease and diagnostic tests, (2) importance of linking general information to concrete self-management actions, and (3) usefulness of the tool as an adjunct to the office visit to prepare for patient-clinician communication. Importantly, these themes were consistent among interviews involving both veterans and clinicians. ConclusionsVeterans and clinicians both thought that the Kidney Score Platform would successfully promote communication and discussion about kidney disease in primary care settings. Tension between using medical terminology that is used regularly by clinicians versus lay terminology to promote CKD awareness was a key challenge, and knowledge of this can inform the development of future CKD educational materials