Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K-specific inhibition.

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.

Automated infection risks assessments (AIRa) for decision-making using a blockchain-based alert system: A case study in a representative building.

Indoor air quality (IAQ) is an important parameter in protecting the occupants of an indoor environment. Previous studies have shown that an indoor environment with poor ventilation increases airborne virus transmission. Existing research has concluded that high ventilation rates can reduce the risk of individuals in indoor environments being infected. However, most existing ventilation systems are designed to be efficient under non-pandemic conditions. Ultimately, indoor environments will become hotspots for the transmission of airborne viruses. Current infection risk assessments can estimate virus transmission via airborne routes, but with limited information sharing among stakeholders. Our own research did not identify any systems that integrate risk assessments with smart sensors in order to support information sharing with experts in indoor environments in their decision-making process. To fill this gap, we designed a blockchain-based prototype (AIRa) that integrates CO2 smart sensor data with infection risk assessments from a post-pandemic perspective. This system generates two types of alerts: (1) P-Alert and (2) R0-Alert for decision-making by building owners, such as increasing the ventilation rate or track and trace, as needed. AIRa shows various benefits over three existing infection-control alert systems. Our solution stores and shares information such as the timestamp and room number, instead of storing building user's personal information. Our approach does not require a QR code to be scanned or a mobile app to be downloaded in order to enable track and trace. However, AIRa is still an early prototype for evaluating the risks of airborne virus transmission in smart building environments. Multidisciplinary knowledge and technological research will be vital in formulating different alerts in the future.

Structural analysis of the LDL receptor-interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site.

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

Reducing Alert Fatigue by Sharing Low-Level Alerts With Patients and Enhancing Collaborative Decision Making Using Blockchain Technology: Scoping Review and Proposed Framework (MedAlert).

BACKGROUND: Clinical decision support (CDS) is a tool that helps clinicians in decision making by generating clinical alerts to supplement their previous knowledge and experience. However, CDS generates a high volume of irrelevant alerts, resulting in alert fatigue among clinicians. Alert fatigue is the mental state of alerts consuming too much time and mental energy, which often results in relevant alerts being overridden unjustifiably, along with clinically irrelevant ones. Consequently, clinicians become less responsive to important alerts, which opens the door to medication errors. OBJECTIVE: This study aims to explore how a blockchain-based solution can reduce alert fatigue through collaborative alert sharing in the health sector, thus improving overall health care quality for both patients and clinicians. METHODS: We have designed a 4-step approach to answer this research question. First, we identified five potential challenges based on the published literature through a scoping review. Second, a framework is designed to reduce alert fatigue by addressing the identified challenges with different digital components. Third, an evaluation is made by comparing MedAlert with other proposed solutions. Finally, the limitations and future work are also discussed. RESULTS: Of the 341 academic papers collected, 8 were selected and analyzed. MedAlert securely distributes low-level (nonlife-threatening) clinical alerts to patients, enabling a collaborative clinical decision. Among the solutions in our framework, Hyperledger (private permissioned blockchain) and BankID (federated digital identity management) have been selected to overcome challenges such as data integrity, user identity, and privacy issues. CONCLUSIONS: MedAlert can reduce alert fatigue by attracting the attention of patients and clinicians, instead of solely reducing the total number of alerts. MedAlert offers other advantages, such as ensuring a higher degree of patient privacy and faster transaction times compared with other frameworks. This framework may not be suitable for elderly patients who are not technology savvy or in-patients. Future work in validating this framework based on real health care scenarios is needed to provide the performance evaluations of MedAlert and thus gain support for the better development of this idea.

Emergency department 'outbreak rostering' to meet challenges of COVID-19.

The COVID-19 outbreak has posed unique challenges to the emergency department rostering. Additional infection control, the possibility of quarantine of staff and minimising contact among staff have significant impact on the work of doctors in the emergency department. Infection of a single healthcare worker may require quarantine of close contacts at work. This may thus affect a potentially large number of staff. As such, we developed an Outbreak Response Roster. This Outbreak Response Roster had fixed teams of doctors working in rotation, each team that staff the emergency department in turn. Members within teams remained constant and were near equally balanced in terms of manpower and seniority of doctors. Each team worked fixed 12 hours shifts with as no overlapping of staff or staggering of shifts. Handovers between shifts were kept as brief as possible. All these were measures to limit interactions among healthcare workers. With the implementation of the roster, measures were also taken to bolster the psychological wellness of healthcare workers. With face-to-face contact limited, we also had to maintain clear, open channels for communication through technology and continue educating residents through innovative means.

Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure.

The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe alphaG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.

Give Intravenous Bolus Overdose a Brake: User Experience and Perception of Safety Device.

OBJECTIVES: Drugs can come in concentrated solutions that require dilution before intravenous bolus administration. Upon dilution, the syringe can contain more than the required amount of drug. The user may mistakenly administer the full contents of the syringe, resulting in an overdose. In this cross-sectional study, we evaluated user experience and perception of Syringe Brake, a dosage flow restrictor device, as part of the intravenous morphine bolus administration workflow. METHODS: From December 2018 to January 2019, doctors and nurses working in the emergency department of 3 public tertiary hospitals in Singapore were invited to complete a paper-based 11-item 5-point Likert scale survey questionnaire after 3 months of Syringe Brake implementation. RESULTS: Overall, 77.5% (290/374; 4.11 ± 0.83) of participants were satisfied with the use of Syringe Brake to prevent medication error. Our survey results showed that the top features of Syringe Brake were ease of setting the desired volume to be administered (86.1%; 4.21 ± 0.72), allowing the drug to be titrated safely (84.8%; 4.26 ± 0.77), and giving users the confidence to avoid overdosing the patient (82.1%; 4.21 ± 0.78). Those with hands-on experience with Syringe Brake rated significantly higher for all survey statements except on the perceived ability to prevent error arising from miscommunication (adjusted odds ratio, 1.58 [0.98-2.57]; P = 0.062). CONCLUSIONS: Syringe Brake shows promising potential for adoption to prevent medication errors. The device serves as a constraint to prevent accidental overdose, caused by user unfamiliarity or autopilot administration.

Reorganising the emergency department to manage the COVID-19 outbreak.

BACKGROUND: The COVID-19 disease outbreak that first surfaced in Wuhan, China, in December 2019, has taken the world by storm and ravaged almost every country in the world. Emergency departments (ED) in hospitals are on the frontlines, serving an essential function in identifying these patients, isolating them early whilst providing urgent medical care. This outbreak has reinforced the role of Emergency Medicine in public health. This paper documents the challenges faced and measures taken by a tertiary hospital's ED in Singapore, in response to the outbreak. MAIN BODY: The ED detected the first case of COVID-19 in Singapore on 22 January 2020 in a Chinese tourist and also the first case of locally transmitted COVID-19 on 3 February 2020. The patient journeys through the patient reception area in the ED and undergoes fever screening before being shunted to isolation areas within the ED. Management and disposition of suspect COVID-19 patients are guided by a close-knit collaboration between ED and department of infectious diseases. With increasing number of patients, back-up plans for expansion of space and staff augmentation have been enacted. Staff safety is also of utmost importance, with provision and guidelines for personal protective equipment and team segregation to ensure no cross-contamination across staff. These have been made possible with an early setup of an operational command and control structure within the ED, managing manpower, logistics, operations, communication and information management and liaison with other clinical departments. CONCLUSION: With the large numbers of undifferentiated patients managed by the ED to date, more than 820 patients with COVID-19 have been identified in the hospital. Not a single member of the staff of the SGH Emergency Department has come down with the illness. The various measures undertaken by the department have helped to ensure good staff morale and strict adherence to safety procedures. We share the lessons learnt so that others who manage EDs around the world can benefit from our experience.

Structural insights into the critical DNA damage sensors DNA-PKcs, ATM and ATR.

ATM, ATR and DNA-PKCs are key effectors of DNA Damage response and have been extensively linked to tumourigenesis and survival of cancer cells after radio/chemotherapy. Despite numerous efforts, the structures of these proteins remained elusive until very recently. The resolution revolution in Cryo-EM allowed for molecular details of these proteins to be seen for the first time. Here we provide a comprehensive review of the structures of ATM, ATR and DNA-PKcs and their complexes and expand with observations springing from our own cryo-EM studies. These observations include a novel conformation of ATR and novel dimeric arrangements of DNA-PKcs.

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase.

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor.

Role of peak current in conversion of patients with ventricular fibrillation.

INTRODUCTION: Peak currents are the final arbiter of defibrillation in patients with ventricular fibrillation (VF). However, biphasic defibrillators continue to use energy in joules for electrical conversion in hopes that their impedance compensation properties will address transthoracic impedance (TTI), which must be overcome when a fixed amount of energy is delivered. However, optimal peak currents for conversion of VF remain unclear. We aimed to determine the role of peak current and optimal peak levels for conversion in collapsed VF patients. METHODS: Adult, non-pregnant patients presenting with non-traumatic VF were included in the study. All defibrillations that occurred were included. Impedance values during defibrillation were used to calculate peak current values. The endpoint was return of spontaneous circulation (ROSC). RESULTS: Of the 197 patients analysed, 105 had ROSC. Characteristics of patients with and without ROSC were comparable. Short duration of collapse < 10 minutes correlated positively with ROSC. Generally, patients with average or high TTI converted at lower peak currents. 25% of patients with high TTI converted at 13.3 ± 2.3 A, 22.7% with average TTI at 18.2 ± 2.5 A and 18.6% with low TTI at 27.0 ± 4.7 A (p = 0.729). Highest peak current conversions were at < 15 A and 15-20 A. Of the 44 patients who achieved first-shock ROSC, 33 (75.0%) received < 20 A peak current vs. > 20 A for the remaining 11 (25%) patients (p = 0.002). CONCLUSION: For best effect, priming biphasic defibrillators to deliver specific peak currents should be considered.

Advances in recombinant protein expression for use in pharmaceutical research.

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells.

X-ray structural analysis of tau-tubulin kinase†1 and its interactions with small molecular inhibitors.

Tau-tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer's disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14-313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co-expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high-affinity ATP-competitive inhibitors, 3-[(6,7-dimethoxyquinazolin-4-yl)amino]phenol (1) and methyl 2-bromo-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzoate (2), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation-primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199-D200 peptide backbone conformation together with altered hydrogen bonding with compound 2. These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer's disease.

Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.