Practical guidance on intensification of insulin therapy with BIAsp 30: a consensus statement

P>Background

Imaging Molecular Structure through Femtosecond Photoelectron Diffraction on Aligned and Oriented Gas-Phase Molecules

This paper gives an account of our progress towards performing femtosecond time-resolved photoelectron diffraction on gas-phase molecules in a pump-probe setup combining optical lasers and an X-ray Free-Electron Laser. We present results of two experiments aimed at measuring photoelectron angular distributions of laser-aligned 1-ethynyl-4-fluorobenzene (C8H5F) and dissociating, laseraligned 1,4-dibromobenzene (C6H4Br2) molecules and discuss them in the larger context of photoelectron diffraction on gas-phase molecules. We also show how the strong nanosecond laser pulse used for adiabatically laser-aligning the molecules influences the measured electron and ion spectra and angular distributions, and discuss how this may affect the outcome of future time-resolved photoelectron diffraction experiments.Comment: 24 pages, 10 figures, Faraday Discussions 17

Anti-interleukin-21 antibody and liraglutide for the preservation of β-cell function in adults with recent-onset type 1 diabetes : a randomised, double-blind, placebo-controlled, phase 2 trial

Publisher Copyright: © 2021 Elsevier LtdBackground: Type 1 diabetes is characterised by progressive loss of functional β-cell mass, necessitating insulin treatment. We aimed to investigate the hypothesis that combining anti-interleukin (IL)-21 antibody (for low-grade and transient immunomodulation) with liraglutide (to improve β-cell function) could enable β-cell survival with a reduced risk of complications compared with traditional immunomodulation. Methods: This randomised, parallel-group, placebo-controlled, double-dummy, double-blind, phase 2 trial was done at 94 sites (university hospitals and medical centres) in 17 countries. Eligible participants were adults aged 18–45 years with recently diagnosed type 1 diabetes and residual β-cell function. Individuals with unstable type 1 diabetes (defined by an episode of severe diabetic ketoacidosis within 2 weeks of enrolment) or active or latent chronic infections were excluded. Participants were randomly assigned (1:1:1:1), with stratification by baseline stimulated peak C-peptide concentration (mixed-meal tolerance test [MMTT]), to the combination of anti-IL-21 and liraglutide, anti-IL-21 alone, liraglutide alone, or placebo, all as an adjunct to insulin. Investigators, participants, and funder personnel were masked throughout the treatment period. The primary outcome was the change in MMTT-stimulated C-peptide concentration at week 54 (end of treatment) relative to baseline, measured via the area under the concentration-time curve (AUC) over a 4 h period for the full analysis set (intention-to-treat population consisting of all participants who were randomly assigned). After treatment cessation, participants were followed up for an additional 26-week off-treatment observation period. This trial is registered with ClinicalTrials.gov, NCT02443155. Findings: Between Nov 10, 2015, and Feb 27, 2019, 553 adults were assessed for eligibility, of whom 308 were randomly assigned to receive either anti-IL-21 plus liraglutide, anti-IL-21, liraglutide, or placebo (77 assigned to each group). Compared with placebo (ratio to baseline 0·61, 39% decrease), the decrease in MMTT-stimulated C-peptide concentration from baseline to week 54 was significantly smaller with combination treatment (0·90, 10% decrease; estimated treatment ratio 1·48, 95% CI 1·16–1·89; p=0·0017), but not with anti-IL-21 alone (1·23, 0·97–1·57; p=0·093) or liraglutide alone (1·12, 0·87–1·42; p=0·38). Despite greater insulin use in the placebo group, the decrease in HbA1c (a key secondary outcome) at week 54 was greater with all active treatments (−0·50 percentage points) than with placebo (−0·10 percentage points), although the differences versus placebo were not significant. The effects diminished upon treatment cessation. Changes in immune cell subsets across groups were transient and mild (<10% change over time). The most frequently reported adverse events included gastrointestinal disorders, in keeping with the known side-effect profile of liraglutide. The rate of hypoglycaemic events did not differ significantly between active treatment groups and placebo, with an exception of a lower rate in the liraglutide group than in the placebo group during the treatment period. No events of diabetic ketoacidosis were observed. One participant died while on liraglutide (considered unlikely to be related to trial treatment) in connection with three reported adverse events (hypoglycaemic coma, pneumonia, and brain oedema). Interpretation: The combination of anti-IL-21 and liraglutide could preserve β-cell function in recently diagnosed type 1 diabetes. The efficacy of this combination appears to be similar to that seen in trials of other disease-modifying interventions in type 1 diabetes, but with a seemingly better safety profile. Efficacy and safety should be further evaluated in a phase 3 trial programme. Funding: Novo Nordisk.Peer reviewe

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 theory1, 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 explodes2. This was first demonstrated on biological samples a decade ago on the giant mimivirus3. Since then a large collaboration4 has been pushing the limit of the smallest sample that can be imaged5,6. 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 diameter7 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

Coherent diffractive imaging of microtubules using an X-ray laser

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature

Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser

A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids

Femtosecond dark-field imaging with an X-ray free electron laser

The emergence of femtosecond diffractive imaging with X-ray lasers has enabled pioneering structural studies of isolated particles, such as viruses, at nanometer length scales. However, the issue of missing low frequency data significantly limits the potential of X-ray lasers to reveal sub-nanometer details of micrometer-sized samples. We have developed a new technique of dark-field coherent diffractive imaging to simultaneously overcome the missing data issue and enable us to harness the unique contrast mechanisms available in dark-field microscopy. Images of airborne particulate matter (soot) up to two microns in length were obtained using single-shot diffraction patterns obtained at the Linac Coherent Light Source, four times the size of objects previously imaged in similar experiments. This technique opens the door to femtosecond diffractive imaging of a wide range of micrometer-sized materials that exhibit irreproducible complexity down to the nanoscale, including airborne particulate matter, small cells, bacteria and gold-labeled biological samples. (C) 2012 Optical Society of Americ

Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser

Citation: Ekeberg, T., Svenda, M., Seibert, M. M., Abergel, C., Maia, F. R. N. C., Seltzer, V., . . . Hajdu, J. (2016). Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser. Scientific Data, 3. doi:10.1038/sdata.2016.60Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms