Attosecond vortex pulse trains

[EN]The landscape of ultrafast structured light pulses has significantly advanced thanks to the ability of high-order harmonic generation (HHG) to translate the spatial properties of infrared laser beams to the extreme-ultraviolet (EUV) spectral range. In particular, the up-conversion of orbital angular momentum (OAM) has enabled the generation of high-order harmonics whose OAM scales linearly with the harmonic order and the topological charge of the driving field. Having a well-defined OAM, each harmonic is emitted as an EUV femtosecond vortex pulse. However, the order-dependent OAM across the harmonic comb precludes the synthesis of attosecond vortex pulses. Here we demonstrate a method for generating attosecond vortex pulse trains, i.e., a succession of attosecond pulses with a helical wavefront, resulting from the coherent superposition of a comb of EUV high-order harmonics with the same OAM. By driving HHG with a polarization tilt-angle fork grating, two spatially separated circularly polarized high-order harmonic beams with order-independent OAM are created. Our work opens the route towards attosecond-resolved light-matter interactions with two extra degrees of freedom, spin and OAM, which are particularly interesting for probing chiral systems and magnetic materials.European Research Council (851201)Ministerio de Ciencia e Innovación (PID2022-142340NB-I00)Air Force Office of Scientific Research (FA9550-22-1-0495

Metabolic Engineering for Biocatalyst Robustness to Organic Inhibitors

Microbial production of biorenewable fuels and chemicals is often limited by inhibition of the biocatalyst, either by increasing concentrations of the product compound or by contaminant compounds in the biomass‐derived sugars. This inhibition can interfere with economically viable production. Here we discuss typical mechanisms of inhibition and methods for improving biocatalyst robustness. Inhibition often takes the form of inhibition of enzyme activity, depletion of cofactor pools, and membrane damage; methods are discussed for mitigating each of these types of inhibition. Various evolutionary schemes have been developed and implemented on a variety of inhibitory compounds, including butanol, acetic acid, furfural, and ethanol. Reverse engineering of these improved strains can provide insight into new metabolic engineering strategies

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.</p

Abstract 925: A genetically engineered mouse model for carcinogenesis by the human enzyme APOBEC3B

Abstract Introduction: Advanced DNA sequencing technologies have revealed a substantial endogenous source of mutations in cancers, the human DNA-mutating enzyme APOBEC3B (A3B). This protein changes DNA cytosines into uracils (C-to-U), which can become “immortalized” in the genome as C-to-T or C-to-G mutations depending on how each uracil lesion is processed. However, mice lack analogous carcinogenic A3 enzymes, which makes a definitive cause-and-effect model of A3-induced carcinogenesis difficult to establish. Results: Using C57BL/6 mice, we have generated a novel genetically engineered mouse model (GEMM) expressing the human protein A3B, where it is driven constitutively from the Rosa26 locus in combination with the strong synthetic CAG promoter. Importantly, tumor-free survival data suggest that CAG A3B mice develop tumors at an increased rate compared to wild-type mice. These CAG A3B mice develop predominantly lymphoid neoplasms including B- and T-cell lymphomas and marked splenomegaly, and less frequently lung and liver cancers. Using immunohistochemical and whole-genome sequencing techniques, we have further characterized the tumors arising in these novel GEMMs. The above analyses thus far have revealed that A3B is actively driving tumor formation, as seen by an enrichment of canonical APOBEC-related mutations in these cancers. Conclusion: Overall, the CAG A3B mice are the first GEMMs demonstrating the potential for A3B to drive tumor formation in vivo, with a predilection towards blood malignancies. They will be an invaluable tool for characterizing A3B-driven mutational processes in an organism and testing novel therapeutic strategies against APOBEC-induced cancer initiation and progression. Citation Format: Cameron C. Durfee, Prokopios P. Argyris, Matthew C. Jarvis, Rena Levin-Klein, Emily K. Law, Reuben S. Harris. A genetically engineered mouse model for carcinogenesis by the human enzyme APOBEC3B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 925.</jats:p

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

AbstractHigh-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3′-to-5′ exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3′ end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.</jats:p

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

Significance SARS-CoV-2 nonstructural protein 14 (nsp14) exoribonuclease (ExoN) plays important roles in the proofreading during viral RNA synthesis and the evasion of host immune responses. We used X-ray crystallography, molecular dynamics simulations, and biochemical assays to investigate the structure, dynamics, and RNA-binding mechanisms of nsp14-ExoN and how its activity is regulated by another viral protein, nsp10. We also demonstrated that nsp14-ExoN can collaborate with the viral RNA polymerase to enable RNA synthesis in the presence of a chain-terminating drug, biochemically recapitulating the proofreading process. Our studies provide mechanistic insights into the functions of a key viral enzyme and a basis for future development of chemical inhibitors.</jats:p

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence

Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an&nbsp;in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity