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The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA–protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic “poisons” perturbs the TOP2 reaction cycle, leading to an accumulation of DNA–protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATT Znf451 -licensed TDP2-catalyzed TOP2-DPC reversal mechanism.

Finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure by cryo-electron microscopy (cryo-EM). While automation has significantly increased the speed of data collection, specimens are still screened manually, a laborious and subjective task that often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-EM. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-EM.

DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3′-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. Here, X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA unveil that LIG1 employs Mg 2+ -reinforced DNA binding to validate DNA base pairing during the adenylyl transfer and nick-sealing ligation reaction steps. Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg 2+ , and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage. DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. Here the authors present X-ray structures and kinetic analyses of LIG1 complexes with undamaged and oxidatively damaged DNA that unveil determinants of LIG1 substrate recognition and enzymatic fidelity.

Coronaviruses use approximately two‐thirds of their 30‐kb genomes to encode nonstructural proteins (nsps) with diverse functions that assist in viral replication and transcription, and evasion of the host immune response. The SARS‐CoV‐2 pandemic has led to renewed interest in the molecular mechanisms used by coronaviruses to infect cells and replicate. Among the 16 Nsps involved in replication and transcription, coronaviruses encode two ribonucleases that process the viral RNA—an exonuclease (Nsp14) and an endonuclease (Nsp15). In this review, we discuss recent structural and biochemical studies of these nucleases and the implications for drug discovery. Coronaviruses encode two ribonucleases: Nsp14, an exonuclease, and Nsp15, an endonuclease. This review covers the latest structures of SARS‐CoV‐2 Nsp14 and Nsp15 and discusses insights into nuclease functions and progress towards therapeutic inhibitors. The viral particle was generated using BioRender.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Propelled by improvements in hardware for data collection and processing, single particle cryo-electron microscopy has rapidly gained relevance in structural biology. Yet, finding the conditions to stabilize a macromolecular target for imaging remains the most critical barrier to determining its structure. Attaining the optimal specimen requires the evaluation of multiple grids in a microscope as conditions are varied. While automation has significantly increased the speed of data collection, optimization is still carried out manually. This laborious process which is highly dependent on subjective assessments, inefficient and prone to error, often determines the success of a project. Here, we present SmartScope, the first framework to streamline, standardize, and automate specimen evaluation in cryo-electron microscopy. SmartScope employs deep-learning-based object detection to identify and classify features suitable for imaging, allowing it to perform thorough specimen screening in a fully automated manner. A web interface provides remote control over the automated operation of the microscope in real time and access to images and annotation tools. Manual annotations can be used to re-train the feature recognition models, leading to improvements in performance. Our automated tool for systematic evaluation of specimens streamlines structure determination and lowers the barrier of adoption for cryo-electron microscopy. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://docs.smartscope.org/

Poly(ADP-ribose), or PAR, is a transient, posttranslational modification catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes. PARPs utilize NAD+ as a substrate to generate PAR for self-attachment (termed automodification) or for attachment to target proteins. PARPs have been implicated in processes including, but not limited to: DNA damage repair, metabolic regulation, and programmed cellular death. There are 17 members of the PARP family of enzymes each characterized by the highly homologous C-terminal ADP-ribose transferase fold (ART) of the CAT domain. In contrast to the homology in this domain, the regulatory domains, such as N-terminal region (NTR) and tryptophan(W)-glycine(G)-arginine(R) (WGR) domain, are much less explored. The lack of identified structural information about PARP regulatory domains leaves a substantial gap in our knowledge. PARPs 1, 2, and 3 exhibit DNA damage-dependent activation and are known as DNA damage response PARPS (DDR-PARPs). During the response to DNA damage, DDR-PARPs recognize DNA breaks and increase the production of PAR. PAR aids in the recruitment of subsequent repair factors to the site of DNA damage. Together, PARP-1 and PARP-2 are essential enzymes, both playing key roles in the DNA damage response, but also in other cellular programs such as gene transcription. Despite extensive characterization of PARP-1, there is limited biochemical and structural analysis of PARP-2, which has a unique domain structure and several distinct cellular roles. Prior to the work presented here, several key aspects of PARP-2 mechanism were not established and thus limited our understanding of PARP-2 function, such as how PARP-2 selectively recognizes DNA repair intermediates and acts within a specific repair pathway. To clarify the role of PARP-2 in DNA repair pathways and the DNA damage response, we have undertaken a structural, biochemical, and cellular investigation of PARP-2. The work presented here has resulted in several novel insights into PARP-2 structure and function. Specifically, we conclude that PARP-2 is selectively activated on 5’phosphorylated DNA breaks, which implicates PARP-2 specific activation just prior to DNA break ligation in the DNA repair process. The data establishes that the NTR is natively disordered, recognizes specific DNA breaks, and requires assembly with other PARP-2 domains for a functional DNA damage recognition response. As a result of this research, it is now known that PARP-2 acts through an allosteric mechanism similar to PARP-1, whereby DNA damage recognition is transmitted to the catalytic domain (CAT) through interdomain communication. Additionally, it is now appreciated that the enzymatic activity of PARP-2 is regulated through the autoinhibitory helical domain (HD), a subdomain of CAT, which locally unfolds upon activation. The unfolding of a region in the HD is a unique allosteric mechanism by which the robust catalytic potential of PARP-2 is tightly regulated. Our comprehensive biochemical and structural approach to study PARP-2 in DNA damage repair further differentiates PARP-2 from other DNA damage-dependent PARPs and leads to a more detailed understanding of the activation mechanism of DDR-PARPs. PARP inhibition is a novel, molecular targeted approach that specifically kills certain cancers with DNA repair deficiencies such as BRCA-1/2 deficient breast and ovarian cancer. This single agent approach, termed “synthetic lethality”, has advanced into several clinical trials leading to the first approved PARP inhibitor, Olaparib (Lynparza™), for advanced ovarian cancers with genetically abnormal BRCA status. Current PARP inhibitors, including Olaparib, bind to the NAD+ binding site that is well conserved across PARPs, and therefore inhibit both PARP-1 and PARP-2 similarly. Overall, this work has presented a foundation to the understanding of DNA damage recognition and activation of PARP-2, having implications for a better understanding of DDR-PARP biology, which could ultimately lead to improved therapeutic options.

Morbid obesity is associated with adverse airway events including desaturation during deep sedation. Prior works have suggested that proprietary high-flow nasal cannula devices generate positive pressure to all airway structures and may be superior to standard (low-flow) nasal cannula for prevention of desaturation. We hypothesized that, at a similar fraction of inspired oxygen (FiO2), use of a High-Flow Nasal Cannula (HFNC) at maximum flow rate would result in a lower incidence of intra-procedural desaturation episodes in morbidly obese patients compared to standard nasal cannula (SNC) during deep sedation with propofol. This is a pragmatic, prospective, randomized clinical trial at one hospital (NCT03148262, UTSW#112016-058). Morbidly obese patients were randomized to HFNC during propofol sedation for colonoscopy. HFNC was performed using maximum flow rates of 60 liters per minute (LPM) and FiO2 of 0.36–0.40, whereas SNC was performed at 4LPM. The primary endpoint was incidence of arterial oxygen desaturation <90% measured by pulse oximetry. At midpoint enrollment the Data Monitoring Committee (DMC) performed a pre-planned O'Brien and Fleming futility test. Patients were randomized to HFNC (n = 28) or SNC (n = 31). Interim analysis of the primary endpoint showed that the desaturation rates in the HFNC group (39.3%) and the SNC group (45.2%) were not significantly different (p = 0.79). The DMC halted the trial at that point due to futility. At similar FiO2, HFNC was not significantly different from SNC for prevention of arterial oxygen desaturation in morbidly obese patients undergoing propofol sedation for colonoscopy. •Morbidly obese patients are at risk for hypoxemia during deep sedation.•The role of high-flow nasal cannula in anesthetic management is unclear.•No difference in desaturation rates found between HFNC and standard cannula groups.•HFNC did not prevent desaturation in morbidly obese patients under deep sedation.

Polymeric films are drug delivery systems that maintain contact with the delivery tissue and sustain a controlled release of therapeutic molecules. These systems allow a longer time of drug contact with the target site in the case of topical treatments and allow the controlled administration of drugs. They can be manufactured by various methods such as solvent casting, hot melt extrusion, electrospinning, and 3D bioprinting. Furthermore, they can employ various polymers, for example PVP, PVA, cellulose derivatives, chitosan, gelling gum, pectin, and alginate. Its versatility is also applicable to different routes of administration, as it can be administered to the skin, oral mucosa, vaginal canal, and eyeballs. All these factors allow numerous combinations to obtain a better treatment. This review focuses on exploring some possible ways to develop them and some particularities and advantages/disadvantages in each case. It also aims to show the versatility of these systems and the advantages and disadvantages in each case, as they bring the opportunity to develop different medicines to facilitate therapies for the most diverse purposes .