Lignin Nanoparticles Deliver Novel Thymine Biomimetic Photo-Adducts with Antimelanoma Activity

We report here the synthesis of novel thymine biomimetic photo-adducts bearing an alkane spacer between nucleobases and characterized by antimelanoma activity against two mutated cancer cell lines overexpressing human Topoisomerase 1 (TOP1), namely SKMEL28 and RPMI7951. Among them, Dewar Valence photo-adducts showed a selectivity index higher than the corresponding pyrimidine-(6-4)-pyrimidone and cyclobutane counterpart and were characterized by the highest affinity towards TOP1/DNA complex as evaluated by molecular docking analysis. The antimelanoma activity of novel photo-adducts was retained after loading into UV photo-protective lignin nanoparticles as stabilizing agent and efficient drug delivery system. Overall, these results support a combined antimelanoma and UV sunscreen strategy involving the use of photo-protective lignin nanoparticles for the controlled release of thymine dimers on the skin followed by their sacrificial transformation into photo-adducts and successive inhibition of melanoma and alert of cellular UV machinery repair pathways.

Continuous B- to A- Transition in Protein-DNA Binding - How Well Is It Described by Current AMBER Force Fields?

When DNA interacts with a protein, its structure often undergoes significant conformational adaptation. Perhaps the most common is the transition from canonical B-DNA towards the A-DNA form, which is not a two-state, but rather a continuous transition. The A- and B- forms differ mainly in sugar pucker P (north/south) and glycosidic torsion χ (high-anti/anti). The combination of A-like P and B-like χ (and vice versa) represents the nature of the intermediate states lying between the pure A- and B- forms. In this work, we study how the A/B equilibrium and in particular the A/B intermediate states, which are known to be over-represented at protein-DNA interfaces, are modeled by current AMBER force fields. Eight protein-DNA complexes and their naked (unbound) DNAs were simulated with OL15 and bsc1 force fields as well as an experimental combination OL15χOL3. We found that while the geometries of the A-like intermediate states in the molecular dynamics (MD) simulations agree well with the native X-ray geometries found in the protein-DNA complexes, their populations (stabilities) are significantly underestimated. Different force fields predict different propensities for A-like states growing in the order OL15 < bsc1 < OL15χOL3, but the overall populations of the A-like form are too low in all of them. Interestingly, the force fields seem to predict the correct sequence-dependent A-form propensity, as they predict larger populations of the A-like form in naked (unbound) DNA in those steps that acquire A-like conformations in protein-DNA complexes. The instability of A-like geometries in current force fields may significantly alter the geometry of the simulated protein-DNA complex, destabilize the binding motif, and reduce the binding energy, suggesting that refinement is needed to improve description of protein-DNA interactions in AMBER force fields.

Constructing DNA logic circuits based on the toehold preemption mechanism

Preemptor blocks the strand displacement reaction by acting on DNA complex, not by directly hybridizing with the worker.

Characterising the transcriptome of hypersegmented human neutrophils

Background: Mature human neutrophils are characterised by their multilobed nuclear morphology. Neutrophil hypersegmentation, a pathologic nuclear phenotype, has been described in the alveolar compartment of patients with acute respiratory distress syndrome and in several other contexts. This study aimed to characterise the transcriptional changes associated with neutrophil hypersegmentation. Methods: A model of hypersegmentation was established by exposing healthy peripheral blood neutrophils to the angiotensin converting enzyme inhibitor (ACEi) captopril. Laser capture microdissection (LCM) was then adapted to isolate a population of hypersegmented neutrophils. Transcriptomic analysis of microdissected hypersegmented neutrophils was undertaken using ribonucleic acid (RNA) sequencing. Differential gene expression (DEG) and enrichment pathway analysis were conducted to investigate the mechanisms underlying hypersegmentation. Results: RNA-Seq analysis revealed the transcriptomic signature of hypersegmented neutrophils, with five genes differentially expressed. VCAN, PADI4 and DUSP4 were downregulated, while LTF and PSMC4 were upregulated. Modulated pathways included histone modification, protein-DNA complex assembly and antimicrobial humoral response. The role of PADI4 was further validated using the small molecule inhibitor, Cl-amidine. Conclusions: Hypersegmented neutrophils display a marked transcriptomic signature, characterised by the differential expression of five genes. This study provides insights into the mechanisms underlying neutrophil hypersegmentation and describes a novel method to isolate and sequence neutrophils based on their morphologic subtype.

A helicase-tethered ORC flip enables bidirectional helicase loading

Replication origins are licensed by loading two Mcm2‑7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires ORC, Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with FRET, we investigated interactions between ORC and Mcm2‑7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2‑7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first and second helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2‑7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly-coordinated series of events through which a single ORC molecule can load two oppositely-oriented helicases.

How the Innate Immune DNA Sensing cGAS–STING Pathway Is Involved in Autophagy

The cGAS–STING pathway is a key component of the innate immune system and exerts crucial roles in the detection of cytosolic DNA and invading pathogens. Accumulating evidence suggests that the intrinsic cGAS–STING pathway not only facilitates the production of type I interferons (IFN-I) and inflammatory responses but also triggers autophagy. Autophagy is a homeostatic process that exerts multiple effects on innate immunity. However, systematic evidence linking the cGAS–STING pathway and autophagy is still lacking. Therefore, one goal of this review is to summarize the known mechanisms of autophagy induced by the cGAS–STING pathway and their consequences. The cGAS–STING pathway can trigger canonical autophagy through liquid-phase separation of the cGAS–DNA complex, interaction of cGAS and Beclin-1, and STING-triggered ER stress–mTOR signaling. Furthermore, both cGAS and STING can induce non-canonical autophagy via LC3-interacting regions and binding with LC3. Subsequently, autophagy induced by the cGAS–STING pathway plays crucial roles in balancing innate immune responses, maintaining intracellular environmental homeostasis, alleviating liver injury, and limiting tumor growth and transformation.

Structural basis for topological regulation of Tn3 resolvase

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest site-specific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely-related resolvases from the Tn3 and γδ transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket – targeting P-pocket by fragment screening

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.

Elevated Myeloperoxidase-DNA Complex Levels in Sera of Patients with IgA Vasculitis

<b><i>Introduction:</i></b> IgA vasculitis is a systemic disease that results from the entrapment of circulating IgA-containing immune complexes in small-vessel walls in the skin, kidneys, and gastrointestinal tract. An excessive formation of neutrophil extracellular traps (NETs) is involved in the pathogenesis of vasculitis, especially in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. This study aimed to clarify whether NETs are implicated in IgA vasculitis. <b><i>Methods:</i></b> Twenty-two patients with IgA vasculitis and 4 healthy volunteers were enrolled in this study. Serum levels of myeloperoxidase (MPO)-DNA complex, a fragment derived from NETs, were determined by enzyme-linked immunosorbent assay (ELISA), and the association between MPO-DNA complex levels and clinical parameters was examined. The presence of the ANCA was also assessed by ELISA specific for MPO and proteinase 3 (PR3) and indirect immunofluorescence (IIF), followed by assessing the differences in clinical parameters with and without the ANCA. <b><i>Results:</i></b> Serum MPO-DNA complex levels were significantly higher in patients with IgA vasculitis than those in healthy controls. A significant positive correlation between the serum MPO-DNA complex and IgA levels was noted. Interestingly, 63.6% of IgA vasculitis patients were ANCA-positive in IIF with an atypical pattern, whereas neither MPO-ANCA nor PR3-ANCA was detected by ELISA. These findings indicated that some IgA vasculitis patients possessed the so called minor ANCA. Serum IgA and MPO-DNA complex levels and the frequency of hematuria in the minor ANCA-positive group were significantly higher than in the minor ANCA-negative group. <b><i>Conclusion:</i></b> The collective findings suggested that NETs are certainly involved in the pathogenesis of IgA vasculitis.