Inter-combination line frequencies in 171Yb validated with the clock transition: erratum.

This erratum corrects an error in Appl. Opt.62, 3932 (2023)APOPAI0003-693510.1364/AO.488653. The correction does not affect the results and conclusions of the original paper.

Intercombination line frequencies in 171Yb validated with the clock transition.

We have carried absolute frequency measurements of the (6s 2) 1 S 0-(6s6p) 3 P 1 transition in 171 Y b (intercombination line), where the spin-1/2 isotope yields two hyperfine lines. The measurements rely on sub-Doppler spectroscopy to yield a discriminator to which a 556 nm laser is locked. The frequency reference for the optical frequency measurements is a high-quality quartz oscillator steered to the GNSS time scale that is bridged with a frequency comb. The reference is validated to ∼3×10-12 by spectroscopy on the 1 S 0- 3 P 0 (clock) line in laser cooled and trapped 171 Y b atoms. From the hyperfine separation between the F=1/2 and F=3/2 levels of 3 P 1, we determine the hyperfine constant to be A(3 P 1)=3957833(28)k H z.

Isolation and characterization of a SARS-CoV-2 variant with a Q677H mutation in the spike protein.

We isolated 20 SARS-CoV-2 strains from positive clinical samples collected in Columbus, Ohio, and investigated the replication of one pair of isolates: a clade 20G strain and a variant of this strain carrying a Q677H mutation in the spike protein and six other amino acid mutations. The OSU.20G variant replicated to a higher peak infectious titer than the 20G base strain in Vero-E6 cells, but the titers were similar when both strains were grown in Calu-3 cells. These results suggest that the OSU.20G variant has increased replication fitness compared to the 20G base strain. This may have contributed to its emergence in December 2020-January 2021.

Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein­drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.

Mutation-Specific SARS-CoV-2 PCR Screen: Rapid and Accurate Detection of Variants of Concern and the Identification of a Newly Emerging Variant with Spike L452R Mutation.

The emergence of more transmissible and/or more virulent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) has triggered intensive genomic surveillance, which is costly and difficult to sustain operationally over the long term. To address this problem, we developed a set of four multiplex mutation-specific PCR-based assays with same-day reporting that can detect five VOC and three variants of interest (VOI), as defined in the March 2021 guidelines from the U.S. Centers for Disease Control and Prevention (https://www.cdc.gov/coronavirus/2019-ncov/). The screening results were compared to the whole-genome sequencing (WGS) and showed 100% concordance for strain typing for B.1.1.7 (n = 25) and P.1 (n = 5) variants using spike (S) mutation S-N501Y, S-E484K, and S-H69-V70del assays. The S-L450R assay, designed to detect the B.1.427/429 VOC, also identified multiple isolates of a newly emerging multiply mutated B.1.526.1 variant that is now rapidly increasing in the eastern United States. PCR approaches can be easily adopted in clinical laboratories, providing rapid screening methods to allow early detection of newly emergent variants and to efficiently triage cases for full genomic sequencing.

MicroRNAs regulate the immunometabolic response to viral infection in the liver.

Immune regulation of cellular metabolism can be responsible for successful responses to invading pathogens. Viruses alter their hosts' cellular metabolism to facilitate infection. Conversely, the innate antiviral responses of mammalian cells target these metabolic pathways to restrict viral propagation. We identified miR-130b and miR-185 as hepatic microRNAs (miRNAs) whose expression is stimulated by 25-hydroxycholesterol (25-HC), an antiviral oxysterol secreted by interferon-stimulated macrophages and dendritic cells, during hepatitis C virus (HCV) infection. However, 25-HC only directly stimulated miR-185 expression, whereas HCV regulated miR-130b expression. Independently, miR-130b and miR-185 inhibited HCV infection. In particular, miR-185 significantly restricted host metabolic pathways crucial to the HCV life cycle. Interestingly, HCV infection decreased miR-185 and miR-130b levels to promote lipid accumulation and counteract 25-HC's antiviral effect. Furthermore, miR-185 can inhibit other viruses through the regulation of immunometabolic pathways. These data establish these microRNAs as a key link between innate defenses and metabolism in the liver.

The ß-Lactamase Assay: Harnessing a FRET Biosensor to Analyse Viral Fusion Mechanisms.

The ß-lactamase (BlaM) assay was first revealed in 1998 and was demonstrated to be a robust Förster resonance energy transfer (FRET)-based reporter system that was compatible with a range of commonly-used cell lines. Today, the BlaM assay is available commercially as a kit and can be utilised readily and inexpensively for an array of experimental procedures that require a fluorescence-based readout. One frequent application of the BlaM assay is the measurement of viral fusion-the moment at which the genetic material harboured within virus particles is released into the cytosol following successful entry. The flexibility of the system permits evaluation of not only total fusion levels, but also the kinetics of fusion. However, significant variation exists in the scientific literature regarding the methodology by which the assay is applied to viral fusion analysis, making comparison between results difficult. In this review we draw attention to the disparity of these methodologies and examine the advantages and disadvantages of each approach. Successful strategies shown to render viruses compatible with BlaM-based analyses are also discussed.

Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. CONCLUSION: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus.

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.

A genetic interaction between the core and NS3 proteins of hepatitis C virus is essential for production of infectious virus.

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1(T)-64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1(T)-64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1(T)-64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.

The utility of implantable loop recorders in patient management: an age- and indication-stratified study in the outpatient-implant era.

AIM: Implantable loop recorders (ILRs) are now routinely implanted for long-term cardiac monitoring in the clinical setting. The aim of this study was to examine the real-world performance of these devices focusing on the management changes made in response to ILR-recorded data. METHODS AND RESULTS: This was a single-centre, prospective observational study of consecutive patients undergoing ILR implantation. All patients who underwent implantation of a Medtronic Reveal LINQ device from September 2017 to June 2019 at Barts Heart Centre were included. Five hundred and one patients were included. Three hundred and two (60%) patients underwent ILR implantation for an indication of pre-syncope/syncope, 96 (19%) for palpitations, 72 (14%) for atrial fibrillation (AF) detection with a history of cryptogenic stroke, and 31 (6%) for high risk of serious cardiac arrhythmia. The primary outcome of this study was that an ILR-derived diagnosis altered management in 110 patients (22%). Secondary outcomes concerned subgroup analyses by indication: in patients who presented with syncope/pre-syncope, a change in management resulting from ILR data was positively associated with age [hazard ratio (HR) 1.04, 95% confidence interval 1.02-1.06; P < 0.001] and negatively associated with a normal electrocardiogram at baseline (HR 0.54 [0.31-0.93]; P = 0.03). Few patients (1/57, 2%) aged <40 years in this group underwent device implantation, compared to 19/62 patients (31%) aged 75 years and over (P = 0.0024). Out of 183 (12%) patients, 22 in the 40-74 age range had a device implanted. Among patients who underwent ILR insertion following cryptogenic stroke, 13/72 (18%) had AF detected, leading to a decision to commence anticoagulation. CONCLUSION: These results inform the utility of ILR in the clinical setting. Diagnoses provided by ILR that lead to changes in management are rare in patients under age 40, particularly following syncope, pre-syncope, or palpitations. In older patients, new diagnoses are frequently made and trigger important changes in treatment.

The Cold-Adapted, Temperature-Sensitive SARS-CoV-2 Strain TS11 Is Attenuated in Syrian Hamsters and a Candidate Attenuated Vaccine.

Live attenuated vaccines (LAVs) replicate in the respiratory/oral mucosa, mimic natural infection, and can induce mucosal and systemic immune responses to the full repertoire of SARS-CoV-2 structural/nonstructural proteins. Generally, LAVs produce broader and more durable protection than current COVID-19 vaccines. We generated a temperature-sensitive (TS) SARS-CoV-2 mutant TS11 via cold-adaptation of the WA1 strain in Vero E6 cells. TS11 replicated at >4 Log10-higher titers at 32 °C than at 39 °C. TS11 has multiple mutations, including those in nsp3, a 12-amino acid-deletion spanning the furin cleavage site of the S protein and a 371-nucleotide-deletion spanning the ORF7b-ORF8 genes. We tested the pathogenicity and protective efficacy of TS11 against challenge with a heterologous virulent SARS-CoV-2 D614G strain 14B in Syrian hamsters. Hamsters were randomly assigned to mock immunization-challenge (Mock-C) and TS11 immunization-challenge (TS11-C) groups. Like the mock group, TS11-vaccinated hamsters did not show any clinical signs and continuously gained body weight. TS11 replicated well in the nasal cavity but poorly in the lungs and caused only mild lesions in the lungs. After challenge, hamsters in the Mock-C group lost weight. In contrast, the animals in the TS11-C group continued gaining weight. The virus titers in the nasal turbinates and lungs of the TS11-C group were significantly lower than those in the Mock-C group, confirming the protective effects of TS11 immunization of hamsters. Histopathological examination demonstrated that animals in the Mock-C group had severe pulmonary lesions and large amounts of viral antigens in the lungs post-challenge; however, the TS11-C group had minimal pathological changes and few viral antigen-positive cells. In summary, the TS11 mutant was attenuated and induced protection against disease after a heterologous SARS-CoV-2 challenge in Syrian hamsters.

Hepatitis C virus: assembly and release of virus particles.

Hepatitis C virus is a blood-borne virus that typically establishes a chronic infection in the liver, which often results in cirrhosis and hepatocellular carcinoma. Progress in understanding the complete virus life cycle has been greatly enhanced by the recent availability of a tissue culture system that produces infectious virus progeny. Thus, it is now possible to gain insight into the roles played by viral components in assembly and egress and the cellular pathways that contribute to virion formation. This minireview describes the key determining viral and host factors that are needed to produce infectious virus.

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication.

Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5'-3' exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.

The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus.

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.

DOACs for stroke prevention in patients with AF and cancer.

Stroke prophylaxis in atrial fibrillation is an important consideration in patients with cancer. However, there is little consensus on the choice of anticoagulation, due to the numerous difficulties associated with active cancer. Direct oral anticoagulants (DOACs) have been shown to be a promising option. Here, we conduct a simple cross-sectional analysis of 29 cancer patients receiving DOACs for stroke prophylaxis in atrial fibrillation at a tertiary-care institution in London. Our study demonstrates an encouraging efficacy and safety profile of DOACs used in this setting. We conclude by suggesting that, while DOACs may be useful, anticoagulation in cancer patients should continue to be individualised.

Metastasis-associated protein 1 transgenic mice: a new model of spontaneous B-cell lymphomas.

Metastasis-associated protein 1 (MTA1), a component of the nuclear remodeling complex and the founding homologue of the MTA family, has been implicated in metastasis, but definitive causative evidence in an animal model system is currently lacking. Here, we show that MTA1 overexpression in transgenic mice is accompanied by a high incidence of spontaneous B cell lymphomas including diffuse large B cell lymphomas (DLBCL). Lymphocytes and lymphoma cells from MTA1-TG mice are hyperproliferative. Lymphomas were transplantable and of clonal origin and were characterized by down-regulation of p27Kip1 as well as up-regulation of Bcl2 and cyclin D1. The significance of these murine studies was established by evidence showing a widespread up-regulation of MTA1 in DLBCL from humans. These findings reveal a previously unrecognized role for the MTA1 pathway in the development of spontaneous B cell lymphomas, and offer a potential therapeutic target in B cell lymphomas. These observations suggest that MTA1-TG mice represent a new model of spontaneous DLBCL associated with high tumor incidence and could be used for therapeutic intervention studies.

MicroRNA-7 mediates cross-talk between metabolic signaling pathways in the liver.

MicroRNAs (miRNAs) have emerged as critical regulators of cellular metabolism. To characterise miRNAs crucial to the maintenance of hepatic lipid homeostasis, we examined the overlap between the miRNA signature associated with inhibition of peroxisome proliferator activated receptor-α (PPAR-α) signaling, a pathway regulating fatty acid metabolism, and the miRNA profile associated with 25-hydroxycholesterol treatment, an oxysterol regulator of sterol regulatory element binding protein (SREBP) and liver X receptor (LXR) signaling. Using this strategy, we identified microRNA-7 (miR-7) as a PPAR-α regulated miRNA, which activates SREBP signaling and promotes hepatocellular lipid accumulation. This is mediated, in part, by suppression of the negative regulator of SREBP signaling: ERLIN2. miR-7 also regulates genes associated with PPAR signaling and sterol metabolism, including liver X receptor ß (LXR-ß), a transcriptional regulator of sterol synthesis, efflux, and excretion. Collectively, our findings highlight miR-7 as a novel mediator of cross-talk between PPAR, SREBP, and LXR signaling pathways in the liver.

Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material.

HIV-1 disseminates to diverse tissues and establishes long-lived viral reservoirs. These reservoirs include the CNS, in which macrophage-lineage cells, and as suggested by many studies, astrocytes, may be infected. Here, we have investigated astrocyte infection by HIV-1. We confirm that astrocytes trap and internalize HIV-1 particles for subsequent release but find no evidence that these particles infect the cell. Astrocyte infection was not observed by cell-free or cell-to-cell routes using diverse approaches, including luciferase and GFP reporter viruses, fixed and live-cell fusion assays, multispectral flow cytometry, and super-resolution imaging. By contrast, we observed intimate interactions between HIV-1-infected macrophages and astrocytes leading to signals that might be mistaken for astrocyte infection using less stringent approaches. These results have implications for HIV-1 infection of the CNS, viral reservoir formation, and antiretroviral therapy.