The Impact of Preprocedural Platelet Function Testing on Periprocedural Complication Rates Associated With Pipeline Flow Diversion: An International Multicenter Study.

BACKGROUND AND OBJECTIVES: Dual antiplatelet therapy (DAPT) is necessary to minimize the risk of periprocedural thromboembolic complications associated with aneurysm embolization using pipeline embolization device (PED). We aimed to assess the impact of platelet function testing (PFT) on reducing periprocedural thromboembolic complications associated with PED flow diversion in patients receiving aspirin and clopidogrel. METHODS: Patients with unruptured intracranial aneurysms requiring PED flow diversion were identified from 13 centers for retrospective evaluation. Clinical variables including the results of PFT before treatment, periprocedural DAPT regimen, and intracranial complications occurring within 72 h of embolization were identified. Complication rates were compared between PFT and non-PFT groups. Differences between groups were tested for statistical significance using the Wilcoxon rank sum, Fisher exact, or χ 2 tests. A P -value <.05 was statistically significant. RESULTS: 580 patients underwent PED embolization with 262 patients dichotomized to the PFT group and 318 patients to the non-PFT group. 13.7% of PFT group patients were clopidogrel nonresponders requiring changes in their pre-embolization DAPT regimen. Five percentage of PFT group [2.8%, 8.5%] patients experienced thromboembolic complications vs 1.6% of patients in the non-PFT group [0.6%, 3.8%] ( P = .019). Two (15.4%) PFT group patients with thromboembolic complications experienced permanent neurological disability vs 4 (80%) non-PFT group patients. 3.7% of PFT group patients [1.5%, 8.2%] and 3.5% [1.8%, 6.3%] of non-PFT group patients experienced hemorrhagic intracranial complications ( P > .9). CONCLUSION: Preprocedural PFT before PED treatment of intracranial aneurysms in patients premedicated with an aspirin and clopidogrel DAPT regimen may not be necessary to significantly reduce the risk of procedure-related intracranial complications.

Periprocedural management of ruptured blister aneurysms treated with pipeline flow diversion.

Background: Blister aneurysms are high-risk intracranial vascular lesions. Definitive treatment of these lesions has been challenging. Severe disability or mortality rates are as high as 55% when these lesions are treated with open surgery. Recent data show that flow diversion is a safe and effective alternative treatment for blister aneurysms. Rerupture of the functionally unsecured lesion remains a concern as flow diversion does not immediately exclude the aneurysm from the circulation. Methods: A retrospective review was performed of any patients with ruptured blister aneurysms treated with a pipeline embolization device between 2010 and 2020 at the University of Colorado. Results: In this paper, we present the results of the intensive care management of ruptured intracranial blister aneurysms after flow-diverting stent placement. Conclusion: Despite the need for dual antiplatelet therapy and the delayed occlusion of blister aneurysms treated with flow diversion, we did not find an increase in periprocedural complications.

Clinical Decision Support for Patients Presenting With Large Vessel Occlusion.

A single center had a collaborative, multidisciplinary review to determine how to best implement new acute ischemic stroke trials involving large vessel occlusions. A flow diagram process map was created for clinical decision support. Patients were divided into four groups based upon size of infarct and timing of presentation. The process map, available in the electronic health record (EHR) for clinicians to reference, guides the selection of patients for endovascular therapy with neuroimaging. In addition, the process map offers guidance for discussions with families and patients experiencing large vessel occlusions with both small and large core infarcts. This manuscript describes the process of creating the process map through a multidisciplinary review and discussion, with points of controversy and how these were addressed.

Enhanced TROSY Effect in [2-19 F, 2-13 C] Adenosine and ATP Analogs Facilitates NMR Spectroscopy of Very Large Biological RNAs in Solution.

Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2-19 F, 2-13 C]-adenosine, which allow the incorporation of aromatic 19 F-13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2-19 F, 2-13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2-19 F, 2-13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2-19 F, 2-13 C]-A-labeled RNA featured sharper resonances than the [2-1 H, 2-13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2-19 F, 2-13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2-19 F, 2-13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.

X-ray Crystallography Module in MD Simulation Program Amber 2023. Refining the Models of Protein Crystals.

The MD simulation package Amber offers an attractive platform to refine crystallographic structures of proteins: (i) state-of-the-art force fields help to regularize protein coordinates and reconstruct the poorly diffracting elements of the structure, such as flexible loops; (ii) MD simulations restrained by the experimental diffraction data provide an effective strategy to optimize structural models of protein crystals, including explicitly modeled interstitial solvent as well as crystal contacts. Here, we present the new crystallography module xray, released as a part of the Amber 2023 package. This module contains functions to calculate and scale structure factors (including the contributions from bulk solvent), evaluate the maximum-likelihood-type crystallographic potential, and compute its derivative forces. The X-ray functionality of Amber no longer relies on external dependencies so that the full advantage of GPU acceleration can be taken. This makes it possible to refine in a short time hundreds of crystal models, including supercell models comprised of multiple unit cells. The new automated Amber-based refinement procedure leads to an appreciable improvement in Rfree (in some cases, by as much as 0.067) as well as MolProbity scores.

An RNA excited conformational state at atomic resolution.

Sparse and short-lived excited RNA conformational states are essential players in cell physiology, disease, and therapeutic development, yet determining their 3D structures remains challenging. Combining mutagenesis, NMR spectroscopy, and computational modeling, we determined the 3D structural ensemble formed by a short-lived (lifetime ~2.1 ms) lowly-populated (~0.4%) conformational state in HIV-1 TAR RNA. Through a strand register shift, the excited conformational state completely remodels the 3D structure of the ground state (RMSD from the ground state = 7.2 ± 0.9 Å), forming a surprisingly more ordered conformational ensemble rich in non-canonical mismatches. The structure impedes the formation of the motifs recognized by Tat and the super elongation complex, explaining why this alternative TAR conformation cannot activate HIV-1 transcription. The ability to determine the 3D structures of fleeting RNA states using the presented methodology holds great promise for our understanding of RNA biology, disease mechanisms, and the development of RNA-targeting therapeutics.

AmberTools.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

The Impact of Dual Antiplatelet Therapy Duration on Unruptured Aneurysm Occlusion After Flow Diversion: A Multicenter Study.

OBJECTIVE: Endoluminal flow diversion reduces blood flow into intracranial aneurysms, promoting thrombosis. Postprocedural dual antiplatelet therapy (DAPT) is necessary for the prevention of thromboembolic complications. The purpose of this study is to therefore assess the impact that the type and duration of DAPT has on aneurysm occlusion rates and iatrogenic complications after flow diversion. METHODS: A retrospective review of a multicenter aneurysm database was performed from 2012 to 2020 to identify unruptured intracranial aneurysms treated with single device flow diversion and ≥12-month follow-up. Clinical and radiologic data were analyzed with aneurysm occlusion as a function of DAPT duration serving as a primary outcome measure. RESULTS: Two hundred five patients underwent flow diversion with a single pipeline embolization device with 12.7% of treated aneurysms remaining nonoccluded during the study period. There were no significant differences in aneurysm morphology or type of DAPT used between occluded and nonoccluded groups. Nonoccluded aneurysms received a longer mean duration of DAPT (9.4 vs 7.1 months, P = 0.016) with a significant effect of DAPT duration on the observed aneurysm occlusion rate (F(2, 202) = 4.2, P = 0.016). There was no significant difference in the rate of complications, including delayed ischemic strokes, observed between patients receiving short (≤6 months) and prolonged duration (>6 months) DAPT (7.9% vs 9.3%, P = 0.76). CONCLUSIONS: After flow diversion, an abbreviated duration of DAPT lasting 6 months may be most appropriate before transitioning to low-dose aspirin monotherapy to promote timely aneurysm occlusion while minimizing thromboembolic complications.

MD simulations of macromolecular crystals: Implications for the analysis of Bragg and diffuse scattering.

Some of our most detailed information about structure and dynamics of macromolecules comes from X-ray-diffraction studies in crystalline environments. More than 170,000 atomic models have been deposited in the Protein Data Bank, and the number of observations (typically of intensities of Bragg diffraction peaks) is generally quite large, when compared to other experimental methods. Nevertheless, the general agreement between calculated and observed intensities is far outside the experimental precision, and the majority of scattered photons fall between the sharp Bragg peaks, and are rarely taken into account. This chapter considers how molecular dynamics simulations can be used to explore the connections between microscopic behavior in a crystalline lattice and observed scattering intensities, and point the way to new atomic models that could more faithfully recapitulate Bragg intensities and extract useful information from the diffuse scattering that lies between those peaks.

New experimental evidence for pervasive dynamics in proteins.

There is ample computational, but only sparse experimental data suggesting that pico-ns motions with 1 Å amplitude are pervasive in proteins in solution. Such motions, if present in reality, must deeply affect protein function and protein entropy. Several NMR relaxation experiments have provided insights into motions of proteins in solution, but they primarily report on azimuthal angle variations of vectors of covalently-linked atoms. As such, these measurements are not sensitive to distance fluctuations, and cannot but under-represent the dynamical properties of proteins. Here we analyze a novel NMR relaxation experiment to measure amide proton transverse relaxation rates in uniformly 15 N labeled proteins, and present results for protein domain GB1 at 283 and 303 K. These relaxation rates depend on fluctuations of dipolar interactions between 1 HN and many nearby protons on both the backbone and sidechains. Importantly, they also report on fluctuations in the distances between these protons. We obtained a large mismatch between rates computed from the crystal structure of GB1 and the experimental rates. But when the relaxation rates were calculated from a 200 ns molecular dynamics trajectory using a novel program suite, we obtained a substantial improvement in the correspondence of experimental and theoretical rates. As such, this work provides novel experimental evidence of widespread motions in proteins. Since the improvements are substantial, but not sufficient, this approach may also present a new benchmark to help improve the theoretical forcefields underlying the molecular dynamics calculations.

Robust total X-ray scattering workflow to study correlated motion of proteins in crystals.

The breathing motions of proteins are thought to play a critical role in function. However, current techniques to study key collective motions are limited to spectroscopy and computation. We present a high-resolution experimental approach based on the total scattering from protein crystals at room temperature (TS/RT-MX) that captures both structure and collective motions. To reveal the scattering signal from protein motions, we present a general workflow that enables robust subtraction of lattice disorder. The workflow introduces two methods: GOODVIBES, a detailed and refinable lattice disorder model based on the rigid-body vibrations of a crystalline elastic network; and DISCOBALL, an independent method of validation that estimates the displacement covariance between proteins in the lattice in real space. Here, we demonstrate the robustness of this workflow and further demonstrate how it can be interfaced with MD simulations towards obtaining high-resolution insight into functionally important protein motions.

Technical Nuances and Outcomes of Telescoping Pipeline Flow Diverters: A Multicenter Study.

BACKGROUND: "Telescoping" multiple overlapping Pipeline Embolization Devices (PEDs; Medtronic) has increased their utility by allowing for more impermeable coverage and providing the ability to off-set landing zone sites and extend treatment constructs. OBJECTIVE: To consider the technical nuances and challenges of telescoping PEDs for the treatment of intracranial aneurysms. METHODS: Databases from 3 U.S. academic neurovascular centers were retrospectively queried to identify patients with intracranial aneurysms treated with multiple PED constructs. Data on patient and aneurysm characteristics, as well as outcomes including Raymond-Roy occlusion classification, modified Rankin Scale score, and complications, were gathered. RESULTS: Forty-six patients had 48 intracranial aneurysms treated, including 16 (33%) in whom placement of telescoping PEDs was planned. Fourteen (30%) patients presented with a ruptured aneurysm. Twenty-one aneurysms (44%) were treated with proximal extension, 13 (27%) with distal extension, and 14 (29%) with PED placement inside one another. Thirty (70%) patients had complete aneurysm occlusion at follow-up. Two (4%) patients had to be retreated. Three patients with unruptured and 1 with ruptured aneurysm had a permanent intraprocedural complication. We present descriptive cases illustrating PEDs that were placed inside one another, proximally, distally, and to improve wall apposition because of vessel tortuosity. CONCLUSION: Our data indicate a higher than expected complication rate that is likely because of the technical complexity of these cases. The case illustrations presented demonstrate the indications and challenging aspects of telescoping PEDs.

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis.

Electrostatic interactions are fundamental to RNA structure and function, and intimately influenced by solvation and the ion atmosphere. RNA enzymes, or ribozymes, are catalytic RNAs that are able to enhance reaction rates over a million-fold, despite having only a limited repertoire of building blocks and available set of chemical functional groups. Ribozyme active sites usually occur at junctions where negatively charged helices come together, and in many cases leverage this strained electrostatic environment to recruit metal ions in solution that can assist in catalysis. Similar strategies have been implicated in related artificially engineered DNA enzymes. Herein, we apply Poisson-Boltzmann, 3D-RISM, and molecular simulations to study a set of metal-dependent small self-cleaving ribozymes (hammerhead, pistol, and Varkud satellite) as well as an artificially engineered DNAzyme (8-17) to examine electrostatic features and their relation to the recruitment of monovalent and divalent metal ions important for activity. We examine several fundamental roles for these ions that include: (1) structural integrity of the catalytically active state, (2) pKa tuning of residues involved in acid-base catalysis, and (3) direct electrostatic stabilization of the transition state via Lewis acid catalysis. Taken together, these examples demonstrate how RNA electrostatics orchestrates the site-specific and territorial binding of metal ions to play important roles in catalysis.

Untangling the Modern Treatment Paradigm for Unruptured Brain Arteriovenous Malformations.

Brain arteriovenous malformations (AVMs) often present treatment challenges. Patients with unruptured AVMs must consider not only whether they want to be treated, but what treatment modality they would prefer. Vascular neurosurgeons, neurointerventional surgeons, and stereotactic radiosurgeons must in turn guide their patients through the most appropriate treatment course considering the risk of AVM rupture, an individual AVM's characteristics, and patient preferences. In this review we will look at how the clinical trial "A Randomized Trial of Unruptured Brain Arteriovenous Malformations (ARUBA)" has influenced the approach to unruptured brain AVMs and the treatment modalities available to clinicians to deal with these formidable lesions.

Tailored Treatment Options for Cerebral Cavernous Malformations.

The diagnosis and treatment of cerebral cavernous malformations (CCMs), or cavernomas, continues to evolve as more data and treatment modalities become available. Intervention is necessary when a lesion causes symptomatic neurologic deficits, seizures, or has high risk of continued hemorrhage. Future medical treatment directions may specifically target the pathogenesis of these lesions. This review highlights the importance of individualized treatment plans based on specific CCM characteristics.

Colicin E1 opens its hinge to plug TolC.

The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins (OMPs) TolC and BtuB to enter the cell. Here, we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryo-electron microscopy to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump, and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other OMP functions.

Bacteria are constantly warring with each other for space and resources. As a result, they have developed a range of molecular weapons to poison, damage or disable other cells. For instance, bacteriocins are proteins that can latch onto structures at the surface of enemy bacteria and push toxins through their outer membrane. Bacteria are increasingly resistant to antibiotics, representing a growing concern for modern healthcare. One way that they are able to survive is by using 'efflux pumps' studded through their external membranes to expel harmful drugs before these can cause damage. Budiardjo et al. wanted to test whether bacteriocins could interfere with this defence mechanism by blocking efflux pumps. Bacteriocins are usually formed of binding elements (which recognise specific target proteins) and of a 'killer tail' that can stab the cell. Experiments showed that the binding parts of a bacteriocin could effectively 'plug' efflux pumps in Escherichia coli bacteria: high-resolution molecular microscopy revealed how the bacteriocin fragment binds to the pump, while fluorescent markers showed that it attached to the surface of E. coli and stopped the efflux pumps from working. As a result, lower amounts of antibiotics were necessary to kill the bacteria when bacteriocins were present. The work by Budiardjo et al. could lead to new ways to combat bacteria that will reduce the need for current antibiotics. In the future, bacteriocins could also be harnessed to target other proteins than efflux pumps, allowing scientists to manipulate a range of bacterial processes.

Integral equation models for solvent in macromolecular crystals.

The solvent can occupy up to ∼70% of macromolecular crystals, and hence, having models that predict solvent distributions in periodic systems could improve the interpretation of crystallographic data. Yet, there are few implicit solvent models applicable to periodic solutes, and crystallographic structures are commonly solved assuming a flat solvent model. Here, we present a newly developed periodic version of the 3D-reference interaction site model (RISM) integral equation method that is able to solve efficiently and describe accurately water and ion distributions in periodic systems; the code can compute accurate gradients that can be used in minimizations or molecular dynamics simulations. The new method includes an extension of the Ornstein-Zernike equation needed to yield charge neutrality for charged solutes, which requires an additional contribution to the excess chemical potential that has not been previously identified; this is an important consideration for nucleic acids or any other charged system where most or all the counter- and co-ions are part of the "disordered" solvent. We present several calculations of proteins, RNAs, and small molecule crystals to show that x-ray scattering intensities and the solvent structure predicted by the periodic 3D-RISM solvent model are in closer agreement with the experiment than are intensities computed using the default flat solvent model in the refmac5 or phenix refinement programs, with the greatest improvement in the 2 to 4 Šrange. Prospects for incorporating integral equation models into crystallographic refinement are discussed.

Molecular dynamics analysis of a flexible loop at the binding interface of the SARS-CoV-2 spike protein receptor-binding domain.

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work, we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.

Determinants of intracranial aneurysm retreatment following embolization with a single flow-diverting stent.

PURPOSE: Flow diverting stents have revolutionized the treatment of intracranial aneurysms through endoluminal reconstruction of the parent vessel. Despite this, certain aneurysms require retreatment. The purpose of this study was to identify clinical and radiologic determinants of aneurysm retreatment following flow diversion. METHODS: A multicenter flow diversion database was evaluated to identify patients presenting with an unruptured, previously untreated aneurysm with a minimum of 12 months' clinical and angiographic follow-up. Univariate and multivariate logistic regression modeling was performed to identify determinants of retreatment. RESULTS: We identified 189 aneurysms treated in 189 patients with a single flow-diverting stent. Mean age was 54 years, and 89% were female. Complete occlusion was achieved in 70.3% and 83.6% of patients at six and 12 months, respectively. Aneurysm retreatment with additional flow-diverting stents occurred in 5.8% of cases. Univariate analysis revealed that dome diameter ≥10 mm (p = 0.012), pre-clinoid internal carotid artery location (p = 0.012), distal > proximal parent vessel diameter (p = 0.042), and later dual antiplatelet therapy (DAPT) discontinuation (p < 0.001) were predictive of retreatment. Multivariate analysis identified discontinuation of DAPT >12 months (p = 0.003) as a strong determinant of retreatment with dome diameter ≥10 mm trending toward statistical significance (p = 0.064). Large aneurysm neck diameter, presence of aneurysm branch vessels, patient age, smoking history, and hypertension were not determinant of retreatment on multivariate analysis. CONCLUSIONS: Prolonged DAPT is the most important determinant of aneurysm retreatment following single-device flow diversion. Abbreviating DAPT duration to only six months should be a consideration in this population, especially for patients with a large aneurysm dome diameter.

Refinement of RNA Structures Using Amber Force Fields.

Atomic models for nucleic acids derived from X-ray diffraction data at low resolution provide much useful information, but the observed scattering intensities can be fit with models that can differ in structural detail. Tradtional geometric restraints favor models that have bond length and angle terms derived from small molecule crystal structures. Here we explore replacing these restraints with energy gradients derived from force fields, including recently developed integral equation models to account for the effects of water molecules and ions that are not part of the explicit model. We compare conventional and force-field based refinements for 22 RNA crystals, ranging in resolution from 1.1 to 3.6 Å. As expected, it can be important to account for solvent screening of charge-charge interactions, especially in the crowded environment of a nucleic acid crystal. The newly refined models can show improvements in torsion angles and hydrogen-bonding interactions, and can significantly reduce unfavorable atomic clashes, while maintaining or improving agreement with observed scattering intensities.