PCR amplification of the positive control.

<p>Note: (a) Amplification results for the cotton endogenous reference gene <i>Sad 1</i>; M: 100-bp marker. (b) Amplification results for the cotton exogenous gene <i>G2</i>-<i>aroA</i>; M: Trans2K^TM DNA marker. 1: ddH₂O; 2: Island cotton 7124; 3: Upland cotton K312; 4: Transgenic cotton BG2-7; 5: Plasmid DNA.</p

Sequences of primers used in this study.

<p>Sequences of primers used in this study.</p

Investigation of Drug-Induced Hepatotoxicity and Its Remediation Pathway with Reaction-Based Fluorescent Probes

Drug-induced liver injury (DILI) is considered a serious problem related to public health, due to its unpredictability and acute response. The level of peroxynitrite (ONOO^–) generated in liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we present two reaction-based fluorescent probes (Naph-ONOO^– and Rhod-ONOO^–) for ONOO^– through a novel and universally applicable mechanism: ONOO^–-mediated deprotection of α-keto caged fluorophores. Among them, Rhod-ONOO^– can selectively accumulate and react in mitochondria, one of the main sources of ONOO^–, with a substantial lower nanomolar sensitivity of 43 nM. The superior selectivity and sensitivity of two probes enable real-time imaging of peroxynitrite generation in lipopolysaccharide-stimulated live cells, with a remarkable difference from cells doped with other interfering reactive oxygen species, in either one- or two-photon imaging modes. More importantly, we elucidated the drug-induced hepatotoxicity pathway with Rhod-ONOO^– and revealed that CYP450/CYP2E1-mediated enzymatic metabolism of acetaminophen leads to ONOO^– generation in liver cells. This is the first time to showcase the drug-induced hepatotoxicity pathways by use of a small-molecule fluorescent probe. We hence conclude that fluorescent probes can engender a deeper understanding of reactive species and their pathological revelations. The reaction-based fluorescent probes will be a potentially useful chemical tool to assay drug-induced hepatotoxicity

Structure of Exogenous Gene Integration and Event-Specific Detection in the Glyphosate-Tolerant Transgenic Cotton Line BG2-7 - Fig 3

<p><b>Amplification results for intraspecific specific qualitative PCR using 5ʹ-F3/5ʹ-R1 (a) and 3ʹ-F6/3ʹ-R12 (b).</b> M: Trans2K^TM DNA marker; 1: ddH₂O; 2: Island cotton 7124; 3: Upland cotton K312; 4: Transgenic cotton BG2-7.</p

A putative integration structure diagram of the exogenous gene insertion.

<p>P-rubisco: Daisy Rubisco small subunit promoter; T-rubisco: Daisy Rubisco small subunit terminator; CTS: Daisy Rubisco small subunit chloroplast signal peptide; P-nos: Nos promoter; T-nos: Nos terminator; <i>npt</i> II: neomycin phosphotransferase gene; <i>G2</i>-<i>aroA</i>: 5-enolpyruvyl-shikimate-3-phosphate synthase gene. Note: The underline represents the missing bases.</p

DNA-Capped Mesoporous Silica Nanoparticles as an Ion-Responsive Release System to Determine the Presence of Mercury in Aqueous Solutions

We have developed DNA-functionalized silica nanoparticles for the rapid, sensitive, and selective detection of mercuric ion (Hg²⁺) in aqueous solution. Two DNA strands were designed to cap the pore of dye-trapped silica nanoparticles. In the presence of ppb level Hg²⁺, the two DNA strands are dehybridized to uncap the pore, releasing the dye cargo with detectable enhancements of fluorescence signal. This method enables rapid (less than 20 min) and sensitive (limit of detection, LOD, 4 ppb) detection, and it was also able to discriminate Hg²⁺ from twelve other environmentally relevant metal ions. The superior properties of the as-designed DNA-functionalized silica nanoparticles can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of thymine-rich DNA with Hg²⁺ . Our design serves as a new prototype for metal-ion sensing systems, and it also has promising potential for detection of various targets in stimulus-release systems

Noncanonical Self-Assembly of Multifunctional DNA Nanoflowers for Biomedical Applications

DNA nanotechnology has been extensively explored to assemble various functional nanostructures for versatile applications. Mediated by Watson–Crick base-pairing, these DNA nanostructures have been conventionally assembled through hybridization of many short DNA building blocks. Here we report the noncanonical self-assembly of multifunctional DNA nanostructures, termed as nanoflowers (NFs), and the versatile biomedical applications. These NFs were assembled from long DNA building blocks generated via rolling circle replication (RCR) of a designer template. NF assembly was driven by liquid crystallization and dense packaging of building blocks, without relying on Watson–Crick base-pairing between DNA strands, thereby avoiding the otherwise conventional complicated DNA sequence design. NF sizes were readily tunable in a wide range, by simply adjusting such parameters as assembly time and template sequences. NFs were exceptionally resistant to nuclease degradation, denaturation, or dissociation at extremely low concentration, presumably resulting from the dense DNA packaging in NFs. The exceptional biostability is critical for biomedical applications. By rational design, NFs can be readily incorporated with myriad functional moieties. All these properties make NFs promising for versatile applications. As a proof-of-principle demonstration, in this study, NFs were integrated with aptamers, bioimaging agents, and drug loading sites, and the resultant multifunctional NFs were demonstrated for selective cancer cell recognition, bioimaging, and targeted anticancer drug delivery

<i>N</i>-Butyl-2-cyanoacrylate-based injectable and <i>in situ</i>-forming implants for efficient intratumoral chemotherapy

<p>The local delivery of chemotherapeutic drugs to tumor sites is an effective approach for achieving therapeutic drug concentrations in solid tumors. Injectable implants with the ability to form <i>in situ</i> represent one of the most promising technologies for intratumoral chemotherapy. However, many issues must be resolved before these implants can be applied in clinical practice. Herein, we report a novel injectable <i>in situ</i>-forming implant system composed of <i>n</i>-butyl-2-cyanoacrylate (NBCA) and ethyl oleate, and the sol–gel phase transition is activated by anions in body fluids or blood. This newly developed injectable NBCA ethyl oleate implant (INEI) is biodegradable, biocompatible, and non-toxic. INEI solidifies in several seconds after exposure to body fluids or blood, and the implant’s <i>in vivo</i> degradation time can be controlled. In addition, the pore sizes formed by the polymerization of NBCA can be decreased by increasing the NBCA concentration in the implants. Therefore, the drug retention/release time can be adjusted from a few weeks to several months by changing the concentration of NBCA in the implant formulation. Anti-tumor experiments in animal models showed that the average growth inhibition rate of xenografted human breast cancer cells by the paclitaxel-loaded INEI (40% NBCA) was 80%, and they also indicated that tumors in some of the mice were completely eliminated by just a single dosage injection. For the epirubicin-loaded INEI (50% NBCA), the average growth inhibition rate of xenografted human liver cancer cells was 58%. Thus, the chemotherapeutic drug-loaded INEIs exhibited excellent therapeutic efficacy for local chemotherapy.</p

Rational Engineering of Bioinspired Anthocyanidin Fluorophores with Excellent Two-Photon Properties for Sensing and Imaging

Fluorescent materials are widely employed in biological analysis owing to their biorthogonal chemistries for imaging and sensing purposes. However, it is always a challenge to design fluorophores with desired photophysical and biological properties, due to their complicated molecular and optical nature. Inspired by anthocyanidin, a class of flower pigments, we designed a new fluorescent molecular framework, AC-Fluor. The new fluorescent materials can be rationally engineered to produce a broad range of fluorescent scaffolds with flexibly tunable emission spectra covering the whole visible light range, from 467 to 707 nm. Furthermore, they exhibit unprecedented environment-insensitive two-photon properties with a substantial cross section as large as 1100 GM in aqueous solution. AC-Fluors demonstrate their biological values through two-photon deep tissue imaging, with penetration depths as much as 300 μm, while exhibiting minimal cytotoxicity. These features engender a rational engineering strategy for the design and optimization of new fluorescent materials for biological imaging

Engineering a Cell-Surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy

Photodynamic therapy is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of selective recognition of cancer cells, controllable activation of photosensitizers, and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on the aptamer can trigger a toehold-mediated catalytic strand displacement to activate the photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine