Heterogeneity in the M. tuberculosis β-Lactamase Inhibition by Sulbactam

15 pags., 7 figs., 1 tab.For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) 1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals 6 . Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating 7-9 , cooperativity, induced fit 10,11 and conformational selection 11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans- enamine. This was made possible in part by the application of the singular value decomposition 14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.This work was supported by NSF-STC-1231306 (BioXFEL). P.F. was supported by NSF BioXFEL STC grant NSF-1231306 Biology with X-ray Lasers, the NIH grant R01GM095583, and the ASU Biodesign Center for Applied Structural Discovery. A.O. was supported by the US Department of Energy, Office of Science, Basic Energy Sciences under award DESC0002164 (underlying dynamical techniques) and by the US National Science Foundation under awards STC-1231306 (underlying data analytical techniques) and DBI-2029533 (underlying analytical models). K.A.Z. was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). D.F, L.A., and E.A.S. were supported by NSF STC BioXFEL center award 6227. L.A. training was supported in part by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) Maximizing Access to Research Careers (MARC) -T34 GM105549 grant. We acknowledge funding from DESY (Hamburg, Germany), a member of the Helmholtz Association HGF; the Cluster of Excellence “Advanced Imaging of Matter” of the Deutsche Forschungsgemeinschaft (DFG) - EXC 2056 - project ID 390715994; the Helmholtz Association Impulse and Networking fund - project InternLabs-0011 “HIR3X”; and the German Federal Ministry of Education and Research (BMBF) - project 05K18CHA. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the U.S. DOE, Office of Science, BES, under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. One or more of the authors of this paper received support from a program designed to increase minority representation in sciencePeer reviewe

Monitoring Temporal Changes in SARS-CoV-2 Spike Antibody Levels and Variant-Specific Risk for Infection, Dominican Republic, March 2021-August 2022

To assess changes in SARS-CoV-2 spike binding antibody prevalence in the Dominican Republic and implications for immunologic protection against variants of concern, we prospectively enrolled 2,300 patients with undifferentiated febrile illnesses in a study during March 2021-August 2022. We tested serum samples for spike antibodies and tested nasopharyngeal samples for acute SARS-CoV-2 infection using a reverse transcription PCR nucleic acid amplification test. Geometric mean spike antibody titers increased from 6.6 (95% CI 5.1-8.7) binding antibody units (BAU)/mL during March-June 2021 to 1,332 (95% CI 1,055-1,682) BAU/mL during May-August 2022. Multivariable binomial odds ratios for acute infection were 0.55 (95% CI 0.40-0.74), 0.38 (95% CI 0.27-0.55), and 0.27 (95% CI 0.18-0.40) for the second, third, and fourth versus the first anti-spike quartile; findings were similar by viral strain. Combining serologic and virologic screening might enable monitoring of discrete population immunologic markers and their implications for emergent variant transmission

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

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Observation of proton-tagged, central (semi)exclusive production of high-mass lepton pairs in pp collisions at 13 TeV with the CMS-TOTEM precision proton spectrometer

The process pp -> pl(+)l(-)p(()*()), with l(+)l(-) a muon or an electron pair produced at midrapidity with mass larger than 110 GeV, has been observed for the first time at the LHC in pp collisions at root s = 13 TeV. One of the two scattered protons is measured in the CMS-TOTEM precision proton spectrometer (CT-PPS), which operated for the first time in 2016. The second proton either remains intact or is excited and then dissociates into a low-mass state p*, which is undetected. The measurement is based on an integrated luminosity of 9.4 fb(-1) collected during standard, high-luminosity LHC operation. A total of 12 mu(+)/mu(-) and 8 e(+)e(-) pairs with m(l(+)l(-)) > 110 GeV, and matching forward proton kinematics, are observed, with expected backgrounds of 1.49 +/- 0.07 (stat) +/- 0.53 (syst) and 2.36 +/- 0.09 (stat) +/- 0.47(syst), respectively. This corresponds to an excess of more than five standard deviations over the expected background. The present result constitutes the first observation of proton-tagged gamma gamma collisions at the electroweak scale. This measurement also demonstrates that CT-PPS performs according to the design specifications.Peer reviewe

Search for strongly interacting massive particles generating trackless jets in proton-proton collisions at s = 13 TeV

A search for dark matter in the form of strongly interacting massive particles (SIMPs) using the CMS detector at the LHC is presented. The SIMPs would be produced in pairs that manifest themselves as pairs of jets without tracks. The energy fraction of jets carried by charged particles is used as a key discriminator to suppress efficiently the large multijet background, and the remaining background is estimated directly from data. The search is performed using proton-proton collision data corresponding to an integrated luminosity of 16.1 fb - 1 , collected with the CMS detector in 2016. No significant excess of events is observed above the expected background. For the simplified dark matter model under consideration, SIMPs with masses up to 100 GeV are excluded and further sensitivity is explored towards higher masses

Measurement of single-diffractive dijet production in proton–proton collisions at s=8 TeV\sqrt{s} = 8\,\text {Te}\text {V} with the CMS and TOTEM experiments

Measurements are presented of the single-diffractive dijet cross section and the diffractive cross section as a function of the proton fractional momentum loss ξ ξ and the four-momentum transfer squared t. Both processes p p → p X p p → p X and p p → X p p p → X p , i.e. with the proton scattering to either side of the interaction point, are measured, where X X includes at least two jets; the results of the two processes are averaged. The analyses are based on data collected simultaneously with the CMS and TOTEM detectors at the LHC in proton–proton collisions at s √ =8TeV s=8TeV during a dedicated run with β ∗ =90m β∗=90m at low instantaneous luminosity and correspond to an integrated luminosity of 37.5nb −1 37.5nb−1 . The single-diffractive dijet cross section σ p X jj σjj p X , in the kinematic region ξ<0.1 ξ<0.1 , 0.03<|t|<1GeV 2 0.03<|t|<1GeV2 , with at least two jets with transverse momentum p T >40GeV pT>40GeV , and pseudorapidity |η|<4.4 |η|<4.4 , is 21.7±0.9(stat) +3.0 −3.3 (syst)±0.9(lumi)nb 21.7±0.9(stat)−3.3+3.0(syst)±0.9(lumi)nb . The ratio of the single-diffractive to inclusive dijet yields, normalised per unit of ξ ξ , is presented as a function of x, the longitudinal momentum fraction of the proton carried by the struck parton. The ratio in the kinematic region defined above, for x values in the range −2.9≤log 10 x≤−1.6 −2.9≤log10⁡x≤−1.6 , is R=(σ p X jj /Δξ)/σ jj =0.025±0.001(stat)±0.003(syst) R=(σjj p X /Δξ)/σjj=0.025±0.001(stat)±0.003(syst) , where σ p X jj σjj p X and σ jj σjj are the single-diffractive and inclusive dijet cross sections, respectively. The results are compared with predictions from models of diffractive and nondiffractive interactions. Monte Carlo predictions based on the HERA diffractive parton distribution functions agree well with the data when corrected for the effect of soft rescattering between the spectator partons