Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry.

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Structural basis for SHOC2 modulation of RAS signalling.

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers1-3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5-8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11-13, but the structure of SHOC2 and the architecture of the presumptive SHOC2-PP1C-RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2-PP1C-MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Core defense hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems.

Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Recent advances in anti-phage system discovery and annotation tools have unearthed many unique systems, often encoded in horizontally transferred defense islands, which can be horizontally transferred. Here, we developed Hidden Markov Models (HMMs) for defense systems and queried microbial genomes on the NCBI database. Out of the 30 species with >200 completely sequenced genomes, our analysis found Pseudomonas aeruginosa exhibits the greatest diversity of anti-phage systems, as measured by Shannon entropy. Using network analysis to identify the common neighbors of anti-phage systems, we identified two core defense hotspot loci (cDHS1 and cDHS2). cDHS1 is up to 224 kb (median: 26 kb) with varied arrangements of more than 30 distinct immune systems across isolates, while cDHS2 has 24 distinct systems (median: 6 kb). Both cDHS regions are occupied in a majority of P. aeruginosa isolates. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identifying core genes flanking immune islands could simplify immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.

Dark Photon Limits from Patchy Dark Screening of the Cosmic Microwave Background.

Dark photons that kinetically mix with the Standard Model photon give rise to new spectral anisotropies (patchy dark screening) in the cosmic microwave background (CMB) due to conversion of photons to dark photons within large-scale structure. We utilize predictions for this patchy dark screening signal to provide the tightest constraints to date on the dark photon kinetic mixing parameter [ϵ≲4.5×10^{-8} (95% confidence level)] over the mass range 10^{-13} eV≲m_{A^{'}}≲10^{-11} eV, almost an order of magnitude stronger than previous limits, by applying state-of-the-art component separation techniques to the cross-correlation of Planck CMB and unWISE galaxy survey data.

Identifying the origins of countertransference using the Core Conflictual Relationship Theme method: A multiple case study approach.

OBJECTIVE: Measurement of countertransference (CT) has proven challenging throughout the history of studying this construct. We sought to determine the potential value of using a common measure of transference, the Core Conflictual Relationship Theme (CCRT) method, as a means of studying CT. METHOD: The Relationship Anecdote Paradigm and the CCRT method were used to examine CT in two studies. In Study 1, we examined the correspondence between a therapist's wishes with significant people in her life (i.e., her parents and husband) and three long-term patients. In Study 2, we identified the interpersonal wishes of a different therapist and examined 14 of her sessions with 3 patients for evidence of how these wishes and needs were displayed in her clinical work. RESULTS: Analyses revealed that specific wishes in therapists' personal lives could be detected from projective interviews and these wishes were similar, but not necessarily identical, to wishes in therapists' descriptions of, and actual work with, their patients. Evidence of both chronic wishes and patient-specific wishes was revealed. CONCLUSIONS: These findings support the idea that the origins of CT reside in therapists' interpersonal wishes and that the CCRT may be a promising means of identifying CT in research, practice, and supervision.

An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7.

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.

Do the Words We Choose Matter When Prescribing Medications?

BACKGROUND: Caregivers are often apprehensive about treating childhood atopic dermatitis (AD) with topical corticosteroids but may find comfort if treatments are presented in a patient-centered manner. OBJECTIVE: We assessed caregivers' willingness to treat AD with either a "topical steroid," "topical medication," or "treatment, similar to the all-natural signals produced by the adrenal glands in the body." METHODS: A survey randomized 874 caregivers of children with AD to receive a "topical steroid," "topical medication," or "treatment, similar to the all-natural signals produced by the adrenal glands in the body." A scenario-only dataset received these descriptions, while a descriptive heading dataset and expanded scale dataset also received headings of "Topical Steroid Use," "Topical Medication Use," and "All-Natural Treatment Use," respectively. Responses were recorded on a 6-point Likert scale or 0-100 slider scale. Whole and dichotomized responses were evaluated using 2-tailed, independent sample t-tests. RESULTS: For the descriptive heading and expanded scale datasets, those presented with a "topical medication" reported greater willingness to treat than those presented with a "topical steroid" and "all-natural treatment" in the descriptive heading dataset (P<0.05). For the dichotomized scenario-only dataset, those presented with a "treatment, similar to the all-natural signals produced by the adrenal glands in the body," reported greater willingness than those presented with a "topical medication" (P<0.05). CONCLUSION: Initially presenting caregivers with a "topical medication" rather than a "topical steroid" may improve willingness to treat AD for some caregivers. However, tailoring the discussion to best fit caregivers’ understanding of treatment may be the most beneficial approach. J Drugs Dermatol. 2023;22(12):1216-1219. doi:10.36849/JDD.5746.

Erratum: New Semiclassical Picture of Vacuum Decay [Phys. Rev. Lett. 123, 031601 (2019)].

This corrects the article DOI: 10.1103/PhysRevLett.123.031601.

The imperfect storm: An uncommon presentation of Chagas disease.

Electrical storm is defined as three or more sustained episodes of ventricular tachycardia, ventricular fibrillation, or appropriate cardioverter-defibrillator shocks during a 24-h period. These patients are notoriously difficult to manage. We present a case secondary to Chagas disease that was responsive to lidocaine. Although an uncommon presentation, given the large-scale population movement from South America, Chagas has an increased incidence and is an important diagnostic consideration in patients with new onset heart failure, arrhythmia, or electrical storm.

First Constraints on Nuclear Coupling of Axionlike Particles from the Binary Neutron Star Gravitational Wave Event GW170817.

Light axion fields, if they exist, can be sourced by neutron stars due to their coupling to nuclear matter, and play a role in binary neutron star mergers. We report on a search for such axions by analyzing the gravitational waves from the binary neutron star inspiral GW170817. We find no evidence of axions in the sampled parameter space. The null result allows us to impose constraints on axions with masses below 10^{-11} eV by excluding the ones with decay constants ranging from 1.6×10^{16} to 10^{18} GeV at a 3σ confidence level. Our analysis provides the first constraints on axions from neutron star inspirals, and rules out a large region in parameter space that has not been probed by the existing experiments.

T cell activation triggers reversible inosine-5'-monophosphate dehydrogenase assembly.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

Assessing the Effect of Clinical Trial Evidence and Anecdote on Caregivers' Willingness to Use Corticosteroids: A Randomized Controlled Trial [Formula: see text].

BACKGROUND: Treatment of childhood atopic dermatitis (AD) is hindered by nonadherence, but caregiver reassurance may help overcome this hurdle. OBJECTIVES: To assess caregivers' willingness to treat childhood AD with a corticosteroid when presented with clinical trial evidence, anecdote, or both. METHODS: A total of 476 caregivers were recruited through a dermatology clinic and online crowdsourcing platform. Subjects were randomized to receive clinical trial evidence, anecdote, or both, using either the term "medication" or "topical steroid." Additional caregivers were queried about their willingness to treat with the doctor's recommendation or without knowledge of its safety information. Responses were recorded on a 10-point Likert scale. RESULTS: Caregivers' willingness to treat was higher in all information assignment groups compared to those not provided with safety information: clinical trial evidence of a "medication" (P = .003; Cohen's d = 0.83) or "topical steroid" (P = .030; d = 0.55), anecdote of a "medication" (P < .0001; d = 1.37) or "topical steroid" (P < .0001; d = 0.85), both clinical trial evidence and anecdote of a "medication" (P < .0001; d = 1.00) or "topical steroid" (P = .000; d = 0.89), and simply the doctor's recommendation (P < .0001; d = 0.92). Significance was corrected for multiple comparisons to 0.0018. There were no differences between caregivers of children with and without AD (P = .36). CONCLUSIONS: Providing anecdotal reassurance, even in the setting of reported high willingness to treat with the doctor's recommendation, may be an effective strategy to improve caregivers' perceptions of starting new medications.

Self-Assembling 2D Arrays with de Novo Protein Building Blocks.

Modular self-assembly of biomolecules in two dimensions (2D) is straightforward with DNA but has been difficult to realize with proteins, due to the lack of modular specificity similar to Watson-Crick base pairing. Here we describe a general approach to design 2D arrays using de novo designed pseudosymmetric protein building blocks. A homodimeric helical bundle was reconnected into a monomeric building block, and the surface was redesigned in Rosetta to enable self-assembly into a 2D array in the C12 layer symmetry group. Two out of ten designed arrays assembled to micrometer scale under negative stain electron microscopy, and displayed the designed lattice geometry with assembly size up to 100 nm under atomic force microscopy. The design of 2D arrays with pseudosymmetric building blocks is an important step toward the design of programmable protein self-assembly via pseudosymmetric patterning of orthogonal binding interfaces.

New Semiclassical Picture of Vacuum Decay.

We introduce a new picture of vacuum decay which, in contrast to existing semiclassical techniques, provides a real-time description and does not rely on classically forbidden tunneling paths. Using lattice simulations, we observe vacuum decay via bubble formation by generating realizations of vacuum fluctuations and evolving with the classical equations of motion. The decay rate obtained from an ensemble of simulations is in excellent agreement with existing techniques. Future applications include bubble correlation functions, fast decay rates, and decay of nonvacuum states.

Transverse Velocities with the Moving Lens Effect.

Gravitational potentials that change in time induce fluctuations in the observed cosmic microwave background (CMB) temperature. Cosmological structure moving transverse to our line of sight provides a specific example known as the moving lens effect. Here, we explore how the observed CMB temperature fluctuations, combined with the observed matter overdensity, can be used to infer the transverse velocity of cosmological structures on large scales. We show that near-future CMB surveys and galaxy surveys will have the statistical power to make a first detection of the moving lens effect, and we discuss applications for the reconstructed transverse velocity.

The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.