Robust Suzuki–Miyaura Cross-Coupling on DNA-Linked Substrates

The Suzuki–Miyaura cross-coupling is one of the most widely employed reactions in medicinal chemistry. To apply this reaction to DNA-encoded library technology (ELT), an alternative approach in the discovery of small molecule hits and leads, we explored the Suzuki–Miyaura cross-coupling on DNA-linked aryl halides. Pd­(PPh₃)₄ was demonstrated to be an effective catalyst for cross-coupling with on-DNA halide substrates under aqueous conditions. It efficiently catalyzes the coupling of phenyl halides (iodide or bromide) and pyridinyl bromides with various boronic acids/esters, including challenging heterocyclic boronic acids/esters

Sequencing the Mouse Genome for the Oxidatively Modified Base 8‑Oxo-7,8-dihydroguanine by OG-Seq

Oxidative damage to the genome can yield the base 8-oxo-7,8-dihydroguanine (OG). In vitro studies suggested OG would preferentially form in 5′-GG-3′ sequence contexts after exposure to reactive oxygen species. Herein, OG locations in the genome were studied by development of “OG-Seq” to sequence OG sites via next-generation sequencing at ∼0.15-kb resolution. The results of this study found ∼10 000 regions of OG enrichment in WT mouse embryonic fibroblasts and ∼18 000 regions when the OG repair glycosylase Ogg1 was knocked out. Gene promoters and UTRs harbor more OG-enriched sites than expected if the sites were randomly distributed throughout the genome and correlate with reactive 5′-GG-3′ sequences, a result supporting decades of in vitro studies. Sequencing of OG paves the way to address chemical and biological questions surrounding this modified DNA base, such as its role in disease-specific mutations and its epigenetic potential in gene regulation

Novel Catalyst System for Suzuki-Miyaura Coupling of Challenging DNA-Linked Aryl Chlorides

A novel Pd catalyst system, [(<i>t</i>-Bu)₂P­(OH)]₂PdCl₂ (POPd) with the ligand sodium 2′-(dicyclohexylphosphino)-2,6-dimethoxy-[1,1′-biphenyl]-3-sulfonate, is reported. It effectively catalyzes the Suzuki-Miyaura coupling of challenging phenyl chlorides and pyrimidinyl chlorides that are covalently linked to a double-stranded DNA-template with various boronic acids/esters

Smartphone Imaging Flow Cytometry for High-Throughput Single-Cell Analysis

We present a portable imaging flow cytometer comprising a smartphone, a small-footprint optical framework, and a PDMS-based microfluidic device. Flow cytometric analysis is performed in a sheathless manner via elasto-inertial focusing with a custom-written Android program, integrating a graphical user interface (GUI) that provides a high degree of user control over image acquisition. The proposed system offers two different operational modes. First, “post-processing” mode enables particle/cell sizing at throughputs of up to 67 000 particles/s. Alternatively, “real-time” mode allows for integrated cell/particle classification with machine learning at throughputs of 100 particles/s. To showcase the efficacy of our platform, polystyrene particles are accurately enumerated within heterogeneous populations using the post-processing mode. In real-time mode, an open-source machine learning algorithm is deployed within a custom-developed Android application to classify samples containing cells of similar size but with different morphologies. The flow cytometer can extract high-resolution bright-field images with a spatial resolution <700 nm using the developed machine learning-based algorithm, achieving classification accuracies of 97% and 93% for Jurkat and EL4 cells, respectively. Our results confirm that the smartphone imaging flow cytometer (sIFC) is capable of both enumerating single particles in flow and identifying morphological features with high resolution and minimal hardware

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (−)-Epigallocatechin Gallate

Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (−)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, <i>P</i> < 0.01). UV–vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound <b>II</b>, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, <i>P</i> < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (<i>P</i> < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (<i>P</i> > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites

Zika Virus Genomic RNA Possesses Conserved G‑Quadruplexes Characteristic of the Flaviviridae Family

Zika virus has emerged as a global concern because neither a vaccine nor antiviral compounds targeting it exist. A structure for the positive-sense RNA genome has not been established, leading us to look for potential G-quadruplex sequences (PQS) in the genome. The analysis identified >60 PQSs in the Zika genome. To minimize the PQS population, conserved sequences in the Flaviviridae family were found by sequence alignment, identifying seven PQSs in the prM, E, NS1, NS3, and NS5 genes. Next, alignment of 78 Zika strain genomes identified a unique PQS near the end of the 3′-UTR. Structural studies on the G-quadruplex sequences found four of the conserved Zika virus sequences to adopt stable, parallel-stranded folds that bind a G-quadruplex-specific compound, and one that was studied caused polymerase stalling when folded to a G-quadruplex. Targeting these PQSs with G-quadruplex binding molecules validated in previous clinical trials may represent a new approach for inhibiting viral replication

Unfolding Kinetics of the Human Telomere i‑Motif Under a 10 pN Force Imposed by the α‑Hemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH

Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the α-hemolysin (α-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (<i>i</i>–<i>t</i>) traces when the i-motif interacts with α-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of α-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5′ 25-mer poly-2′-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the α-HL nanopore, a feature that is not easily achievable by other methods

A Novel Approach toward Polyfulvene: Cationic Polymerization of Enediynes

Enediyne compounds have found limited applications in polymer science and material chemistry due to the poor regioselectivity and/or the step-growth nature in their radical polymerizations. However, the cationic cyclization of enediynes exhibits a high regioselective 5-exo-dig mechanism, providing a new strategy for the synthesis of polyfulvene derivatives. The expected polymers were successfully produced by cationic polymerization of enediynes induced by CF₃SO₃H, and a well-defined conjugated structure was confirmed by NMR, IR, and UV–vis spectroscopy. GPC analysis shows a relatively narrow molecular weight distribution, and the molecular weight reaches up to 62.9 kDa. On the other hand, the structural features of the obtained polymers and the mechanism of the cationic polymerization were investigated through kinetic study and MALDI-TOF MS analysis, which revealed a second-order consumption of enediyne monomer and the polymerization being probably terminated through intramolecular abstraction of proton from the neighboring group

Internal vs Fishhook Hairpin DNA: Unzipping Locations and Mechanisms in the α‑Hemolysin Nanopore

Studies on the interaction of hairpin DNA with the α-hemolysin (α-HL) nanopore have determined hairpin unzipping kinetics, thermodynamics, and sequence-dependent DNA/protein interactions. Missing from these results is a systematic study comparing the unzipping process for fishhook (one-tail) vs internal (two-tail) hairpins when they are electrophoretically driven from the <i>cis</i> to the <i>trans</i> side of α-HL via a 30-mer single-stranded tail. In the current studies, fishhook hairpins showed long unzipping times with one deep blockage current level. In contrast, the internal hairpins demonstrated relatively fast unzipping and a characteristic pulse-like current pattern. These differences were further explored with respect to stem length and sequence context. Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins. Interestingly, these studies were able to resolve a current difference of ∼6% between hairpin DNA immobilized in the nanopore waiting to unzip vs the translocating unzipped DNA, with the latter showing a deeper current blockage level. This demonstration of different currents for immobilized and translocating DNA has not been described previously. These results were interpreted as fishhook hairpins unzipping inside the vestibule, while the internal hairpins unzip outside the vestibule of α-HL. Lastly, we used this knowledge to study the unzipping of a long double-stranded DNA (>50 base pairs) outside the vestibule of α-HL. The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids

Estradiol dose-dependently upregulated Nav1.7 expression in TG and reduced the mechanical threshold in ovariectomized rats.

<p>(A) Plasma level of 17β-estradiol after estrogen replacement. (B) Head withdrawal threshold after estrogen replacement. (C) Nav1.7 mRNA expression in TG after estrogen replacement (n = 4, two-way ANOVA). (D) Representative immunoblotting for Nav1.7 expression in TG after estrogen replacement. β-Actin was served as an internal control for equal loading (n = 3). *<i>P</i> < 0.05 versus control group; ^#<i>P</i> < 0.05 versus 0 μg group; ^&<i>P</i> < 0.05 versus 20 μg group. Data are expressed as mean ± SD.</p