Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.

Implications of inhibition of Rev1 interaction with Y family DNA polymerases for cisplatin chemotherapy.

Chemotherapy with cisplatin becomes limiting due to toxicity and secondary malignancies. In principle, therapeutics could be improved by targeting translesion synthesis (TLS) polymerases (Pols) that promote replication through intrastrand cross-links, the major cisplatin-induced DNA adduct. However, to specifically target malignancies with minimal adverse effects on normal cells, a good understanding of TLS mechanisms in normal versus cancer cells is paramount. We show that in normal cells, TLS through cisplatin intrastrand cross-links is promoted by Polη- or Polι-dependent pathways, both of which require Rev1 as a scaffolding component. In contrast, cancer cells require Rev1-Polζ. Our findings that a recently identified Rev1 inhibitor, JH-RE-06, purported to specifically disrupt Rev1 interaction with Polζ to block TLS through cisplatin adducts in cancer cells, abrogates Rev1's ability to function with Y family Pols as well, implying that by inactivating Rev1-dependent TLS in normal cells, this inhibitor will exacerbate the toxicity and tumorigenicity of chemotherapeutics with cisplatin.

Pyrimidine inhibitors synergize with nucleoside analogues to block SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes2. Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England.

The SARS-CoV-2 lineage B.1.1.7, designated variant of concern (VOC) 202012/01 by Public Health England1, was first identified in the UK in late summer to early autumn 20202. Whole-genome SARS-CoV-2 sequence data collected from community-based diagnostic testing for COVID-19 show an extremely rapid expansion of the B.1.1.7 lineage during autumn 2020, suggesting that it has a selective advantage. Here we show that changes in VOC frequency inferred from genetic data correspond closely to changes inferred by S gene target failures (SGTF) in community-based diagnostic PCR testing. Analysis of trends in SGTF and non-SGTF case numbers in local areas across England shows that B.1.1.7 has higher transmissibility than non-VOC lineages, even if it has a different latent period or generation time. The SGTF data indicate a transient shift in the age composition of reported cases, with cases of B.1.1.7 including a larger share of under 20-year-olds than non-VOC cases. We estimated time-varying reproduction numbers for B.1.1.7 and co-circulating lineages using SGTF and genomic data. The best-supported models did not indicate a substantial difference in VOC transmissibility among different age groups, but all analyses agreed that B.1.1.7 has a substantial transmission advantage over other lineages, with a 50% to 100% higher reproduction number.

DNA polymerase θ accomplishes translesion synthesis opposite 1,N6-ethenodeoxyadenosine with a remarkably high fidelity in human cells.

Here we show that translesion synthesis (TLS) opposite 1,N6-ethenodeoxyadenosine (εdA), which disrupts Watson-Crick base pairing, occurs via Polι/Polζ-, Rev1-, and Polθ-dependent pathways. The requirement of Polι/Polζ is consistent with the ability of Polι to incorporate nucleotide opposite εdA by Hoogsteen base pairing and of Polζ to extend synthesis. Rev1 polymerase and Polθ conduct TLS opposite εdA via alternative error-prone pathways. Strikingly, in contrast to extremely error-prone TLS opposite εdA by purified Polθ, it performs predominantly error-free TLS in human cells. Reconfiguration of the active site opposite εdA would provide Polθ the proficiency for error-free TLS in human cells.

Shapeshifting bullvalene-linked vancomycin dimers as effective antibiotics against multidrug-resistant gram-positive bacteria.

The alarming rise in superbugs that are resistant to drugs of last resort, including vancomycin-resistant enterococci and staphylococci, has become a significant global health hazard. Here, we report the click chemistry synthesis of an unprecedented class of shapeshifting vancomycin dimers (SVDs) that display potent activity against bacteria that are resistant to the parent drug, including the ESKAPE pathogens, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as vancomycin-resistant S. aureus (VRSA). The shapeshifting modality of the dimers is powered by a triazole-linked bullvalene core, exploiting the dynamic covalent rearrangements of the fluxional carbon cage and creating ligands with the capacity to inhibit bacterial cell wall biosynthesis. The new shapeshifting antibiotics are not disadvantaged by the common mechanism of vancomycin resistance resulting from the alteration of the C-terminal dipeptide with the corresponding d-Ala-d-Lac depsipeptide. Further, evidence suggests that the shapeshifting ligands destabilize the complex formed between the flippase MurJ and lipid II, implying the potential for a new mode of action for polyvalent glycopeptides. The SVDs show little propensity for acquired resistance by enterococci, suggesting that this new class of shapeshifting antibiotic will display durable antimicrobial activity not prone to rapidly acquired clinical resistance.

How Global Collaboration Can Improve the Medical Countermeasure Life Cycle for Infectious Disease Outbreaks: A BARDA Perspective.

Infectious disease outbreaks have become increasingly common and require global partnership for adequate preparedness and response. During outbreaks, medical countermeasures (MCMs)-vaccines, therapeutics, and diagnostics-need to reach patients quickly. BARDA utilizes public-private partnerships to support advanced development of MCMs through U.S. FDA approval against a variety of threats within its mission space. MCM preparedness and response must be approached as an integrated life cycle, not as independent steps. Recent filovirus outbreaks in Africa exemplify that collaborative relationships are critical for emergency response, and products with regulatory approval can expand access and reach patients quicker than investigational products. Unfortunately, insufficient funding globally and differences in funders' prioritization puts gains and future efforts at risk. Of primary concern is a) lack of a feasible regulatory path and clinical capability to achieve regulatory approval for new MCMs for many diseases; and b) the need for partners with the mandate, funding, and capabilities to support the life cycle activities following development-long-term sustainment of manufacturing capability and stockpiling of licensed products to support international outbreaks. Finding partners that complement BARDA's mission and support the MCM life cycle will be a key component in deciding which MCM development efforts can be supported. Without collaboration, the global community runs the risk of losing the capabilities built through years of investment and being underprepared to combat future threats. Synergies between funders that have different roles and responsibilities within the MCM life cycle are critical to MCM availability and create long-term sustainment of products to ensure access.

DNA polymerase ε leading strand signature mutations result from defects in its proofreading activity.

The evidence that purified pol2-M644G DNA polymerase (Pol)ε exhibits a highly elevated bias for forming T:dTTP mispairs over A:dATP mispairs and that yeast cells harboring this Polε mutation accumulate A > T signature mutations in the leading strand have been used to assign a role for Polε in replicating the leading strand. Here, we determine whether A > T signature mutations result from defects in Polε proofreading activity by analyzing their rate in Polε proofreading defective pol2-4 and pol2-M644G cells. Since purified pol2-4 Polε exhibits no bias for T:dTTP mispair formation, A > T mutations are expected to occur at a much lower rate in pol2-4 than in pol2-M644G cells if Polε replicated the leading strand. Instead, we find that the rate of A > T signature mutations are as highly elevated in pol2-4 cells as in pol2-M644G cells; furthermore, the highly elevated rate of A > T signature mutations is severely curtailed in the absence of PCNA ubiquitination or Polζ in both the pol2-M644G and pol2-4 strains. Altogether, our evidence supports the conclusion that the leading strand A > T signature mutations derive from defects in Polε proofreading activity and not from the role of Polε as a leading strand replicase, and it conforms with the genetic evidence for a major role of Polδ in replication of both the DNA strands.

Mismatch repair operates at the replication fork in direct competition with mismatch extension by DNA polymerase δ.

DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase δ (Polδ) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Polδ proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Polδ and Polε. Our finding that suppression of elevated mutagenesis in pol3-01 by the Polδ pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Polδ. Furthermore, the evidence that Polδ pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Polδ in replication of both the leading and lagging DNA strands.

Project NexGen: Developing the Next Generation of COVID-19 Vaccines and Therapeutics to Respond to the Present and Prepare for the Future.

COVID-19 epidemiology and product landscapes have changed considerably since onset of the pandemic. Safe and effective vaccines and therapeutics are available, but the continual emergence of SARS-CoV-2 variants introduce limitations in our ability to prevent and treat disease. Project NextGen is a collaboration between the Biomedical Advanced Research and Development Authority (BARDA), part of the Administration for Strategic Preparedness and Response (ASPR), and the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, that is leveraging public-private partnerships to address gaps in the nation's COVID-19 vaccine and therapeutic capabilities. Targeted investments will advance promising next-generation candidates through the most difficult phases of clinical development to encourage further private sector interest for later stage development and commercial availability. New commercial vaccines and therapeutics that are more durable and effective across variants will improve our fight against COVID-19 and transform our response to future threats.

The Detection of Trace Metal Contaminants in Organic Products Using Ion Current Rectifying Quartz Nanopipettes.

While ion current rectification (ICR) in aprotic solvent has been fundamentally studied, its application in sensing devices lacks exploration. The development of sensors operable in these solvents is highly beneficial to the chemical industry, where polar aprotic solvents, such as acetonitrile, are widely used. Currently, this industry relies on the use of inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (OES) for the detection of metal contamination in organic products. Herein, we present the detection of trace amounts of Pd2+ and Co2+ using ion current rectification, in cyclam-functionalized quartz nanopipettes, with tetraethylammonium tetrafluoroborate (TEATFB) in MeCN as supporting electrolyte. This methodology is employed to determine the concentration of Pd in organic products, before and after purification by Celite filtration and column chromatography, obtaining comparable results to ICP-MS within minutes and without complex sample preparation. Finite element simulations are used to support our experimental findings, which reveal that the formation of double-junction diodes in the nanopore enables trace detection of these metals, with a significant response from baseline even at picomolar concentrations.

Diet-induced obesity and diabetes enhance mortality and reduce vaccine efficacy for SARS-CoV-2.

IMPORTANCE: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a wide spectrum of diseases in the human population, from asymptomatic infections to death. It is important to study the host differences that may alter the pathogenesis of this virus. One clinical finding in coronavirus disease 2019 (COVID-19) patients is that people with obesity or diabetes are at increased risk of severe illness from SARS-CoV-2 infection. We used a high-fat diet model in mice to study the effects of obesity and type 2 diabetes on SARS-CoV-2 infection as well as how these comorbidities alter the response to vaccination. We find that diabetic/obese mice have increased disease after SARS-CoV-2 infection and they have slower clearance of the virus. We find that the lungs of these mice have increased neutrophils and that removing these neutrophils protects diabetic/obese mice from disease. This demonstrates why these diseases have increased risk of severe disease and suggests specific interventions upon infection.

Reproducibility and stability of silane layers in nanoconfined electrochemical systems.

Organosilanes are commonly utilized to attach bioreceptors to oxide surfaces. The deposition of such silane layers is especially challenging in nanoscale or nanoconfined devices, such as in nanopipettes, since rinsing off loosely bound silanes may not be possible due to geometric constrictions and because the thickness of multilayered silanes can cover or block nanoscale features. Furthermore, in electrochemical devices, the silane layers experience additional perturbations, such as electric migration and electroosmotic force. Despite its importance, there appears to be no consensus in the current literature on the optimal methodology for nanopipette silanization, with significant variations in reported conditions. Herein, we systematically investigate the reproducibility and stability of liquid- and vapor-phase deposited silane layers inside nanopipettes. Electrochemical monitoring of the changing internal silanized surface reveals that vapor-deposited APTES generates surface modifications with the highest reproducibility, while vapor-deposited APTMS generates surface modifications of the highest stability over a 24-hour time period. Practical issues of silanizing nanoconfined systems are highlighted, and the importance of carefully chosen silanization conditions to yield stable and reproducible monolayers is emphasized as an underappreciated aspect in the development of novel nanoscale systems.

Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.

A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.

Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis.

CLINICAL AND STRUCTURAL IMPACT OF SUBMACULAR FLUID AFTER PNEUMATIC RETINOPEXY REPAIR FOR RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To assess the impact of submacular fluid (SMF) after pneumatic retinopexy for primary rhegmatogenous retinal detachment repair. METHODS: Retrospective review of consecutive patients treated with pneumatic retinopexy for primary rhegmatogenous retinal detachment repair. 387 eyes (374 patients) were included, of which 166 underwent optical coherence tomography imaging after successful pneumatic retinopexy. Foveal-centered optical coherence tomography scans were reviewed. RESULTS: SMF occurred in 59 eyes (35.5%) and was associated with macular detachment ( P ≤ 0.001) and phakic lens status ( P = 0.007). Submacular fluid resolved over an average of 9.39 months and was associated with worse preprocedure best-corrected visual acuity and delayed visual recovery. The mean final best-corrected visual acuity was 0.277 logarithm of the minimum angle of resolution (20/40) in eyes with SMF and 0.162 logarithm of the minimum angle of resolution (20/30) in those without SMF ( P < 0.001). Submacular fluid was associated with discontinuity of the interdigitation zone ( P = 0.003), ellipsoid zone ( P = 0.005), and external limiting membrane ( P ≤ 0.001) after SMF resolution. Ellipsoid zone discontinuity was associated with worse visual prognosis ( P = 0.009). CONCLUSION: Trace SMF detected by optical coherence tomography is common after successful pneumatic retinopexy and resulted in delayed visual recovery and increased rates of outer retinal discontinuity after SMF resorption, although the final difference in best-corrected visual acuity in those with and without SMF was minimal.

Optical and physical properties of annealed amorphous niobium oxide thin films.

The physical and optical properties of coatings deposited using physical vapor deposition techniques exhibit changes during post deposition annealing. Optical properties including the index of refraction and spectral transmission vary when coatings are annealed. Physical and mechanical properties such as thickness, density, and stress are also impacted by annealing. In this paper, we study the source of these changes by examining the impact of 150-500°C annealing on N b 2 O 5 films deposited using thermal evaporation and reactive magnetron sputtering. Models based on the Lorentz-Lorenz equation and potential energy considerations explain the data, and rationalize conflicts between previously reported results.

Prevalence and Severity Distribution of Type 2 Inflammation-Related Comorbidities Among Patients with Asthma, Chronic Rhinosinusitis with Nasal Polyps, and Atopic Dermatitis.

This observational study assessed the prevalence of co-existing type 2 inflammatory conditions [T2Cs; asthma, atopic dermatitis (AD), allergic rhinitis, and chronic rhinosinusitis with nasal polyps (CRSwNP)] in patients with moderate-to-severe (M/S) type 2 asthma, M/S CRSwNP, or M/S AD, in the real-world setting. Data from 761 physicians in the US and EUR5 were sourced from Adelphi Disease-Specific Programmes covering patients with M/S asthma (n = 899), M/S CRSwNP (n = 683), and M/S AD (n = 1497). At least one T2C was identified in 66%, 69%, and 46% of M/S asthma, M/S CRSwNP, and M/S AD cohorts, respectively, and 24%, 36% and 16% had at least two T2Cs; trends were similar in the US and EUR5. In patients with M/S asthma or M/S CRSwNP, T2Cs commonly presented as mild or moderate. The comorbidity burden suggests that an integrated treatment approach is warranted to address underlying type 2 inflammation in patients with M/S type 2 diseases.