An Allosteric Switch Primes Sequence-Specific DNA Recognition.

Regulation of protein-DNA binding specificity occurs through myriad mechanisms. Boudet et al. discover yet a new form of specificity through allosteric regulation, an ATP-induced structural switch that mediates specific DNA recognition in an archaeoeukaryotic primase.

De Novo Human Angiotensin-Converting Enzyme 2 Decoy NL-CVX1 Protects Mice From Severe Disease After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

The emergence of novel variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need to investigate alternative approaches to prevent infection and treat patients with coronavirus disease 2019. Here, we report the preclinical efficacy of NL-CVX1, a de novo decoy that blocks virus entry into cells by binding with nanomolar affinity and high specificity to the receptor-binding domain of the SARS-CoV-2 spike protein. Using a transgenic mouse model of SARS-CoV-2 infection, we showed that a single prophylactic intranasal dose of NL-CVX1 conferred complete protection from severe disease following SARS-CoV-2 infection. Multiple therapeutic administrations of NL-CVX1 also protected mice from succumbing to infection. Finally, we showed that infected mice treated with NL-CVX1 developed both anti-SARS-CoV-2 antibodies and memory T cells and were protected against reinfection a month after treatment. Overall, these observations suggest NL-CVX1 is a promising therapeutic candidate for preventing and treating severe SARS-CoV-2 infections.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

How the onset of the Covid-19 pandemic impacted pro-social behaviour and individual preferences: Experimental evidence from China.

We present experimental evidence on how pro-sociality, trust and attitudes towards risk and ambiguity evolved over the six weeks following the imposition of stringent Covid-19 related lockdown measures in the Hubei province of China. We compare incentivized economic decision-making in a baseline sample, collected pre-epidemic, with a series of repeated cross-sectional samples drawn from the same population between January and March, 2020. We find high rates of altruism, cooperation and aversion to risk taking under ambiguity in the immediate aftermath of the lockdown, while trust is significantly below its baseline level. Risk attitudes also differ in the post-lockdown sample, with decreased risk tolerance in the loss domain and lesser risk aversion in the gain domain. We further uncover significant transitory effects for trust and risk aversion around the date of a high-profile whistleblower's death from Covid-19. Our findings suggest that the onset of a public health crisis may have unintended consequences for economic preferences that determine population compliance with interventions designed to reduce the spread of a novel coronavirus.

Higher 2-Year Cumulative Incidence of Mental Health Disorders Following Irrigation and Debridement in Primary Lumbar Fusion.

Introduction: Spinal fusion is an operation that is employed to treat spinal diseases. Surgical site infection (SSI) after lumbar fusion (LF) is a postoperative complication. SSI is treated with irrigation and debridement (I&D), which requires readmittance following discharge or prolonged hospital stays, which are deleterious to patients' mental health. The long-term relationship between treating SSI with I&D and patients' mental health is still understudied. Methods: Using the Mariner dataset from the PearlDiver Patient Records Database using Current Procedural Terminology and International Classification of Diseases procedure codes, retrospective cohort analysis was carried out. This study involved 445,480 patients who underwent LF with at least 2-year follow-up and were followed up for 2 years. Of the patients, 2,762 underwent I&D. Using univariate analysis employing Pearson Chi-square and Student t-test, where appropriate (Table 1), patient demographics between cohorts were gathered. 2-year cumulative incidence (CI) between LF and I&D cohorts was calculated using Kaplan-Meier analysis (Fig. 1, 2, 3). Cox proportional hazards were employed to observe significant differences in CI rates (Table 2). Results: For patients who received I&D, 2-year CI depression (HR: 1.72; 95% CI: 1.49-1.99; P<0.001) and stress (HR: 1.35; 95% CI: 1.02-1.79; P=0.035) rates were significantly higher than for those who did not. There was no statistically significant difference in 2-year CI anxiety rates between cohorts (HR: 0.92; 95% CI: 0.58-1.46; P=0.719). Conclusions: In conclusion, 16.8% of patients developed new-onset depression 2 years following I&D, in comparison to 10.3% of those who underwent LF. Patients who underwent I&D following LF were significantly more likely to experience depression and stress. To mitigate negative mental health outcomes, mental health services should be available to patients who underwent surgery.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

A Slow Dynamic RNA Switch Regulates Processing of microRNA-21.

The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of many eukaryotic genes, and whose dysregulation is a driver of human disease. Here we report the discovery of a very slow (0.1 s-1) conformational rearrangement at the Dicer cleavage site of pre-miR-21, which regulates the relative concentration of readily- and inefficiently-processed RNA structural states. We show that this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of their unprocessed or processed states.

Structure of the RNA Specialized Translation Initiation Element that Recruits eIF3 to the 5'-UTR of c-Jun.

Specialized translation initiation is a novel form of regulation of protein synthesis, whereby RNA structures within the 5'-UTR regulate translation rates of specific mRNAs. Similar to internal ribosome entry sites (IRESs), specialized translation initiation requires the recruitment of eukaryotic initiation factor 3 (eIF3), but also requires cap recognition by eIF3d, a new 5'-m7GTP recognizing protein. How these RNA structures mediate eIF3 recruitment to affect translation of specific mRNAs remains unclear. Here, we report the nuclear magnetic resonance (NMR) structure of a stem-loop within the c-JUN 5' UTR recognized by eIF3 and essential for specialized translation initiation of this well-known oncogene. The structure exhibits similarity to eIF3 recognizing motifs found in hepatitis C virus (HCV)-like IRESs, suggesting mechanistic similarities. This work establishes the RNA structural features involved in c-JUN specialized translation initiation and provides a basis to search for small molecule inhibitors of aberrant expression of the proto-oncogenic c-JUN.

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2.

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins (∼18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2.

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

Prospectively gated axial CT coronary angiography: preliminary experiences with a novel low-dose technique.

To assess image quality and radiation exposure with prospectively gated axial CT coronary angiography (PGA) compared to retrospectively gated helical techniques (RGH). Forty patients with suspected coronary artery disease (CAD) and a stable heart rate below 65 bpm underwent CT coronary angiography (CTCA) using a 64-channel CT system. The patient cohort consisted of 20 consecutive patients examined using a PGA technique and 20 patients examined using a standard RGH technique. Both groups were matched demographically according to age, gender, body mass index, and heart rate. For both groups, two independent observers assessed image quality for all coronary segments on an ordinal scale from 1 (nonassessable) to 5 (excellent quality). Image quality and radiation exposure were compared between patient groups. There were no significant differences in vessel-based image quality between the two groups (P > 0.05). Mean (+/- SD) effective radiation exposure in the PGA group was 3.7 +/- 0.8 mSv compared to 18.9 +/- 3.8 mSv in the RGH group without ECG-based tube current modulation (P < 0.001). Preliminary experience shows PGA technique to be a promising approach for CTCA resulting in a substantial reduction in radiation exposure with image quality comparable to that of standard RGH technique.

Design of RNA-targeting macrocyclic peptides.

RNA structures play a pivotal role in many biological processes and the progression of human disease, making them an attractive target for therapeutic development. Often RNA structures operate through the formation of complexes with RNA-binding proteins, however, much like protein-protein interactions, RNA-protein interactions span large surface areas and often lack traditional druggable properties, making it challenging to target them with small molecules. Peptides provide much greater surface areas and therefore greater potential for forming specific and high affinity interactions with RNA. In this chapter, we discuss our approach for engineering peptides that bind to structured RNAs by highlighting methods and design strategies from previous successful projects aimed at inhibiting the HIV Tat-TAR interaction and the biogenesis of oncogenic microRNAs.

Standardised Warfarin Reversal Expedites Time to Theatre for Fractured Neck of Femur Surgery and Improves Mortality Rates: A Matched Cohort Study.

BACKGROUND: This study aims to evaluate outcomes for warfarinised hip fracture patients and compare them with a matched nonwarfarinised group, before and after the introduction of national hip fracture guidelines in the United Kingdom. METHODS: A retrospective cohort study of 1743 hip fracture patients was undertaken. All patients admitted taking warfarin were identified. These patients were then matched to nonwarfarinised patients using nearest neighbour propensity score matching, accounting for age, sex, hip fracture type, and Nottingham Hip Fracture Score. A pre-guideline group (no standardised warfarin reversal regimen) and a post-guideline group (standardised regimen) were identified. Outcomes assessed included time to INR less than 1.7, time to theatre, length of stay, and 30-day and 1-year mortality. RESULTS: Forty-six warfarinised hip fracture patients were admitted in the pre-guideline group (mean age 80.5, F:M 3:1) and 48 in the post-guideline group (mean age 81.2 years, F:M 3:1). Post-guideline patients were reversed to a safe operative INR level within 18 hours of admission, decreasing the time to first dose vitamin K (p<0.001). 70% of warfarinised patients were operated upon within 36 hours, compared to 19.6% with no regimen (p<0.05). After anticoagulation reversal protocol, thirty-day mortality decreased from 15.2% to 8.3% and 1-year mortality from 43.5% to 33% for warfarinised patients, which is comparable to nonwarfarinised matched patients. There was no significant change in the length of stay pre- and post-guideline for both groups of patients. CONCLUSIONS: Proactive anticoagulant management and expedient surgery reduces morbidity and mortality when managing this surgically challenging subset of hip fracture patients.

Empirical regioselectivity models for human cytochromes P450 3A4, 2D6, and 2C9.

Cytochromes P450 3A4, 2D6, and 2C9 metabolize a large fraction of drugs. Knowing where these enzymes will preferentially oxidize a molecule, the regioselectivity, allows medicinal chemists to plan how best to block its metabolism. We present QSAR-based regioselectivity models for these enzymes calibrated against compiled literature data of drugs and drug-like compounds. These models are purely empirical and use only the structures of the substrates, in contrast to those models that simulate a specific mechanism like hydrogen radical abstraction, and/or use explicit models of active sites. Our most predictive models use three substructure descriptors and two physical property descriptors. Descriptor importances from the random forest QSAR method show that other factors than the immediate chemical environment and the accessibility of the hydrogen affect regioselectivity in all three isoforms. The cross-validated predictions of the models are compared to predictions from our earlier mechanistic model (Singh et al. J. Med. Chem. 2003, 46, 1330-1336) and predictions from MetaSite (Cruciani et al. J. Med. Chem. 2005, 48, 6970-6979).

A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21 Precursor and Suppresses Dicer Processing.

MicroRNAs (miRNAs) help orchestrate cellular growth and survival through post-transcriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects and chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers, leading to the down-regulation of key tumor-suppressing and pro-apoptotic factors, suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic ß-hairpin peptidomimetics which bind to RNA stem-loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with KD = 200 nM and prefers it over other pre-miRNAs. The NMR structure of the complex shows that the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rationale for the peptide binding activity and clear guidance for further improvements in affinity and targeting.

Cardiac imaging using multislice computed tomography scanners: technical considerations.

Conventional coronary angiography is currently the gold standard in the detection and diagnosis of coronary artery disease. This modality, however, is invasive in nature. Hence, there is a need for noninvasive imaging techniques to provide comprehensive assessment of coronary artery disease, especially in stable patients at low to moderate risk of disease. In recent years, a number of noninvasive modalities have found wide applications in cardiac imaging. Most recent investigations have used magnetic resonance imaging, multislice computed tomography and electron-beam computed tomography scanners. This review discusses the clinical challenges existing in the field of cardiac imaging and focuses on the technical advancements of multislice computed tomography scanners that have made them a very attractive noninvasive option for the detection and diagnosis of coronary artery disease.

Automatic determination of differential coronary artery motion minima for cardiac computed tomography optimal phase selection.

RATIONALE AND OBJECTIVES: Selecting the optimal phase for coronary artery evaluation can be challenging, especially at higher heart rates, given that the optimal phase may differ for each of the coronary arteries. This study aimed to evaluate a novel vessel-specific algorithm which automatically outputs the minimum motion phase per coronary artery. MATERIALS AND METHODS: The study included 44 patients who underwent 256-slice cardiac computed tomography for evaluation of chest pain. End-systolic and mid-diastolic minimal motion phases were automatically calculated by a previously validated global motion algorithm and by a new vessel-specific algorithm which calculates the minimum motion for each of the three main coronary arteries, separately. Two readers blindly evaluated all coronary segments for image quality. Median scores per coronary artery were compared by the Wilcoxon signed rank test. RESULTS: The variation, per patient, between the optimal phases of the three coronary arteries was 5.0 ± 4.5% (1%-22%) for end systole and 4.8 ± 4.1% (0%-19%) for mid diastole. The mean image quality scores per coronary artery were 4.0 ± 0.61 for the vessel-specific approach and 3.80 ± 0.69 for the global phase selection (P < .001). Overall, 46 of 122 arteries had a better score with the vessel-specific approach and five with the standard global approach. Interreader agreement was substantial (k = 0.72). CONCLUSIONS: This study has shown that multiple phases are required to ensure optimal image quality for all three coronary arteries and that a vessel-specific phase selection algorithm achieves superior results to the standard global approach.

A model for predicting likely sites of CYP3A4-mediated metabolism on drug-like molecules.

We have developed a rapid semiquantitative model for evaluating the relative susceptibilities of different sites on drug molecules to metabolism by cytochrome P450 3A4. The model is based on the energy necessary to remove a hydrogen radical from each site, plus the surface area exposure of the hydrogen atom. The energy of hydrogen radical abstraction is conventionally measured by AM1 semiempirical molecular orbital calculations. AM1 calculations show the following order of radical stabilities for the hydrogen atom abstractions: sp2 centers > heteroatom sp3 centers > carbon sp3 centers. Since AM1 calculations are too time intensive for routine work, we developed a statistical trend vector model, which is used to estimate the AM1 abstraction energy of a hydrogen atom from its local atomic environment. We carried out AM1 and trend vector calculations on 50 CYP3A4 substrates whose major sites of metabolism are known in the literature. A plot of the lowest hydrogen radical formation energy versus its sterically accessible surface area exposure for these 50 substrates shows that only those hydrogen atoms with solvent accessible surface area exposure > or = 8.0 A(2) are susceptible to CYP3A4-mediated metabolism. This approach forms the basis for our general model, which predicts sites on drugs that are susceptible to cytochrome P450 3A4-mediated hydrogen radical abstraction followed by a hydroxylation reaction. This model, in conjunction with specific enzyme site binding requirements, can aid in identifying possible sites of metabolism catalyzed by other cytochrome P450 enzymes.

Quantification of aortic valve area at 256-slice computed tomography: comparison with transesophageal echocardiography and cardiac catheterization in subjects with high-grade aortic valve stenosis prior to percutaneous valve replacement.

PURPOSE: The purpose of this study was to compare planimetric aortic valve area (AVA) measurements from 256-slice CT to those derived from transesophageal echocardiography (TEE) and cardiac catheterization in high-risk subjects with known high-grade calcified aortic stenosis. METHODS AND MATERIALS: The study included 26 subjects (10 males, mean age: 79±6; range, 61-88 years). All subjects were clinically referred for aortic valve imaging prior to percutaneous aortic valve replacement from April 2008 to March 2009. Two radiologists, blinded to the results of TEE and cardiac catheterization, independently selected the systolic cardiac phase of maximum aortic valve area and independently performed manual CT AVA planimetry for all subjects. Repeated AVA measurements were made to establish CT intra- and interobserver repeatability. In addition, the image quality of the aortic valve was rated by both observers. Aortic valve calcification was also quantified. RESULTS: All 26 subjects had a high-grade aortic valve stenosis (systolic opening area <1.0 cm(2)) via CT-based planimetry, with a mean AVA of 0.62±0.18. In four subjects, TEE planimetry was precluded due to severe aortic valve calcification, but CT-planimetry was successfully performed with a mean AVA of 0.46±0.23 cm(2). Mean aortic valve calcium mass score was 563.8±526.2 mg. Aortic valve area by CT was not correlated with aortic valve calcium mass score. A bias and limits of agreement among CT and TEE, CT and cardiac catheterization, and TEE and cardiac catheterization were -0.07 [-0.37 to 0.24], 0.03 [-0.49 to 0.55], 0.12 [-0.39 to 0.63]cm(2), respectively. Differences in AVA among CT and TEE or cardiac catheterization did not differ systematically over the range of measurements and were not correlated with aortic valve calcium mass score. CONCLUSION: Planimetric aortic valve area measurements from 256-slice CT agree well with those derived from TEE and cardiac catheterization in high-risk subjects with known high-grade calcified aortic stenosis.

Segmentation of the heart and great vessels in CT images using a model-based adaptation framework.

Recently, model-based methods for the automatic segmentation of the heart chambers have been proposed. An important application of these methods is the characterization of the heart function. Heart models are, however, increasingly used for interventional guidance making it necessary to also extract the attached great vessels. It is, for instance, important to extract the left atrium and the proximal part of the pulmonary veins to support guidance of ablation procedures for atrial fibrillation treatment. For cardiac resynchronization therapy, a heart model including the coronary sinus is needed. We present a heart model comprising the four heart chambers and the attached great vessels. By assigning individual linear transformations to the heart chambers and to short tubular segments building the great vessels, variable sizes of the heart chambers and bending of the vessels can be described in a consistent way. A configurable algorithmic framework that we call adaptation engine matches the heart model automatically to cardiac CT angiography images in a multi-stage process. First, the heart is detected using a Generalized Hough Transformation. Subsequently, the heart chambers are adapted. This stage uses parametric as well as deformable mesh adaptation techniques. In the final stage, segments of the large vascular structures are successively activated and adapted. To optimize the computational performance, the adaptation engine can vary the mesh resolution and freeze already adapted mesh parts. The data used for validation were independent from the data used for model-building. Ground truth segmentations were generated for 37 CT data sets reconstructed at several cardiac phases from 17 patients. Segmentation errors were assessed for anatomical sub-structures resulting in a mean surface-to-surface error ranging 0.50-0.82mm for the heart chambers and 0.60-1.32mm for the parts of the great vessels visible in the images.