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COVID-19 vaccination, risk-compensatory behaviours, and contacts in the UK.
The physiological effects of vaccination against SARS-CoV-2 (COVID-19) are well documented, yet the behavioural effects not well known. Risk compensation suggests that gains in personal safety, as a result of vaccination, are offset by increases in risky behaviour, such as socialising, commuting and working outside the home. This is potentially important because transmission of SARS-CoV-2 is driven by contacts, which could be amplified by vaccine-related risk compensation. Here, we show that behaviours were overall unrelated to personal vaccination, but-adjusting for variation in mitigation policies-were responsive to the level of vaccination in the wider population: individuals in the UK were risk compensating when rates of vaccination were rising. This effect was observed across four nations of the UK, each of which varied policies autonomously.
An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts antiviral T cell activity.
The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.
Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease.
The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.
Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.
Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.
Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function
Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nϵ-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nϵ-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.
An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome
Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5′ truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.
Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.
Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.
DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation.
Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.
Omicron-associated changes in SARS-CoV-2 symptoms in the United Kingdom
Abstract Background The SARS-CoV-2 Delta variant has been replaced by the highly transmissible Omicron BA.1 variant, and subsequently by Omicron BA.2. It is important to understand how these changes in dominant variants affect reported symptoms, while also accounting for symptoms arising from other co-circulating respiratory viruses. Methods In a nationally representative UK community study, the COVID-19 Infection Survey, we investigated symptoms in PCR-positive infection episodes vs. PCR-negative study visits over calendar time, by age and vaccination status, comparing periods when the Delta, Omicron BA.1 and BA.2 variants were dominant. Results Between October-2020 and April-2022, 120,995 SARS-CoV-2 PCR-positive episodes occurred in 115,886 participants, with 70,683 (58%) reporting symptoms. The comparator comprised 4,766,366 PCR-negative study visits (483,894 participants); 203,422 (4%) reporting symptoms. Symptom reporting in PCR-positives varied over time, with a marked reduction in loss of taste/smell as Omicron BA.1 dominated, maintained with BA.2 (44%/45% 17 October 2021, 16%/13% 2 January 2022, 15%/12% 27 March 2022). Cough, fever, shortness of breath, myalgia, fatigue/weakness and headache also decreased after Omicron BA.1 dominated, but sore throat increased, the latter to a greater degree than concurrent increases in PCR-negatives. Fatigue/weakness increased again after BA.2 dominated, although to a similar degree to concurrent increases in PCR-negatives. Symptoms were consistently more common in adults aged 18-65 years than in children or older adults. Conclusions Increases in sore throat (also common in the general community), and a marked reduction in loss of taste/smell, make Omicron harder to detect with symptom-based testing algorithms, with implications for institutional and national testing policies.
Data from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<div>AbstractPurpose:<p>DNA polymerase theta (Polθ, encoded by the <i>POLQ</i> gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy.</p>Experimental Design:<p>We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models <i>in vitro</i> and <i>in vivo</i>.</p>Results:<p>Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. <i>In vivo</i>, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone.</p>Conclusions:<p>These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.</p></div>
Supplementary Figure S6 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Accompanies Figure 5 (ART899 combined with radiation causes significant tumor growth delay in vivo and is well tolerated)</p>
Supplementary Table S1 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Luciferase-based MMEJ assay in a panel of cancer cell lines.</p>
Supplementary Figure S2 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Cell cycle-dependent effects of ART558 in combination with IR</p>
Supplementary Figure S4 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Accompanies Figure 3 (Effect of ART558 under hypoxic conditions)</p>
Supplementary Table S1 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Luciferase-based MMEJ assay in a panel of cancer cell lines.</p>
Supplementary Figure S3 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Accompanies Figure 2 (ART558 treatment leads to increased residual IR-induced DNA damage foci)</p>
Supplementary Figure S5 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Accompanies Figure 4 (Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability)</p>
Supplementary Figure S5 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Accompanies Figure 4 (Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability)</p>
Supplementary Figure S1 from Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models
<p>Distribution of POLQ mRNA levels across cell lines from CCLE</p>