Pd-Catalyzed Cyclization and Carbene Migratory Insertion: New Approach to 3‑Vinylindoles and 3‑Vinylbenzofurans

A Pd-catalyzed stereoselective synthesis of 3-vinylindoles and 3-vinylbenzofurans has been developed. The reaction merges the alkyne-based Pd-catalyzed cyclization and Pd carbene migratory insertion in a single catalytic cycle, generating a C–C single bond and a CC double bond in one operation

Pd-Catalyzed Cyclization and Carbene Migratory Insertion: New Approach to 3‑Vinylindoles and 3‑Vinylbenzofurans

A Pd-catalyzed stereoselective synthesis of 3-vinylindoles and 3-vinylbenzofurans has been developed. The reaction merges the alkyne-based Pd-catalyzed cyclization and Pd carbene migratory insertion in a single catalytic cycle, generating a C–C single bond and a CC double bond in one operation

Photosensitizer-Encapsulated Ferritins Mediate Photodynamic Therapy against Cancer-Associated Fibroblasts and Improve Tumor Accumulation of Nanoparticles

Nanoparticles have been widely tested as drug delivery carriers or imaging agents, largely because of their ability to selectively accumulate in tumors through the enhanced permeability and retention (EPR) effect. However, studies show that many tumors afford a less efficient EPR effect and that many nanoparticles are trapped in the perivascular region after extravasation and barely migrate into tumor centers. This is to a large degree attributed to the dense tumor extracellular matrix (ECM), which functions as a physical barrier to prevent efficient nanoparticle extravasation and diffusion. In this study, we report a photodynamic therapy (PDT) approach to enhance tumor uptake of nanoparticles. Briefly, we encapsulate ZnF₁₆Pc, a photosensitizer, into ferritin nanocages, and then conjugate to the surface of the ferritin a single chain viable fragment (scFv) sequence specific to fibroblast activation protein (FAP). FAP is a plasma surface protein widely upregulated in cancer-associated fibroblasts (CAFs), which is a major source of the ECM fiber components. We found that the scFv-conjugated and ZnF₁₆Pc-loaded ferritin nanoparticles (scFv-Z@FRT) can mediate efficient and selective PDT, leading to eradication of CAFs in tumors. When tested in bilateral 4T1 tumor models, we found that the tumor accumulation of serum albumin (BSA), 10 nm quantum dots (QDs), and 50 nm QDs was increased by 2-, 3.5-, and 18-fold after scFv-Z@FRT mediated PDT. Our studies suggest a novel and safe method to enhance the delivery of nanoparticles to tumors

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted Photoimmunotherapy To Enhance Cytotoxic T Cell Infiltration and Tumor Control

Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle-based photoimmunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier, and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photoirradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor-bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C–X–C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer

Overexpression of the Wheat Aquaporin Gene, <em>TaAQP7</em>, Enhances Drought Tolerance in Transgenic Tobacco

<div><p>Aquaporin (AQP) proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of <em>AQPs</em> in drought stress response is not completely understood in plants. In this study, a <em>PIP2</em> subgroup gene <em>AQP</em>, designated as <em>TaAQP7</em>, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in <em>Xenopus laevis</em> oocytes and <em>TaAQP7</em> transcript was induced by dehydration, and treatments with polyethylene glycol (PEG), abscisic acid (ABA) and H₂O₂. Further, <em>TaAQP7</em> was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of <em>TaAQP7</em> after PEG treatment. Overexpression of <em>TaAQP7</em> increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA) and H₂O₂, and less ion leakage (IL), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities when compared with the wild type (WT) under drought stress. Taken together, our results show that <em>TaAQP7</em> confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants.</p> </div

Genome-Wide Identification and Analysis of MAPK and MAPKK Gene Families in <em>Brachypodium distachyon</em>

<div><p>MAPK cascades are universal signal transduction modules and play important roles in plant growth, development and in response to a variety of biotic and abiotic stresses. Although MAPKs and MAPKKs have been systematically investigated in several plant species including <em>Arabidopsis</em>, rice and poplar, no systematic analysis has been conducted in the emerging monocot model plant <em>Brachypodium distachyon</em>. In the present study, a total of 16 MAPK genes and 12 MAPKK genes were identified from <em>B. distachyon</em>. An analysis of the genomic evolution showed that both tandem and segment duplications contributed significantly to the expansion of MAPK and MAPKK families. Evolutionary relationships within subfamilies were supported by exon-intron organizations and the architectures of conserved protein motifs. Synteny analysis between <em>B. distachyon</em> and the other two plant species of rice and <em>Arabidopsis</em> showed that only one homolog of <em>B. distachyon</em> MAPKs was found in the corresponding syntenic blocks of <em>Arabidopsis</em>, while 13 homologs of <em>B. distachyon</em> MAPKs and MAPKKs were found in that of rice, which was consistent with the speciation process of the three species. In addition, several interactive protein pairs between the two families in <em>B. distachyon</em> were found through yeast two hybrid assay, whereas their orthologs of a pair in <em>Arabidopsis</em> and other plant species were not found to interact with each other. Finally, expression studies of closely related family members among <em>B. distachyon</em>, <em>Arabidopsis</em> and rice showed that even recently duplicated representatives may fulfill different functions and be involved in different signal pathways. Taken together, our data would provide a foundation for evolutionary and functional characterization of MAPK and MAPKK gene families in <em>B. distachyon</em> and other plant species to unravel their biological roles.</p> </div

Osmotic tolerance analysis of <i>TaAQP7</i>-overexpressing plants.

<p>A total of 200 surface-sterilized seeds of each transgenic line, WT or VC germinated on MS medium containing 0 (A, a) or 300 mM (B, b) mannitol for 12 d, and the germination rate was calculated. A, B are photos of the first 12 days after germination on mediums. The WT, VC and transgenic lines were cultured in MS medium under a 16 h light/8 h dark cycle at 25°C for one week, and then the seedlings were transplanted to MS or MS supplied with 150 or 300 mM mannitol for one week. The photographs were taken (C, D, E) and root length was calculated (F). Vertical bars indicate ±SE calculated from four replicates. Asterisks indicate significant difference between the WT and the three transgenic lines (*<i>p</i><0.05; **<i>p</i><0.01). Three biological experiments were performed, which produced similar results.</p

Phylogenetic and domain analyses of MAPKKs.

<p>(a) Phylogenetic relationships of <i>B. distachyon</i>, <i>Arabidopsis</i> and rice MAPKK genes. The phylogenetic tree was constructed by NJ (Neighbor–joining) method using the MEGA 4 program. Different color patternings indicate different gene clusters or superclusters. (b) Schematic diagram of amino acid motifs of <i>B. distachyon</i> MAPKKs. The black solid line represents the corresponding BdMAPKK and its length. The different-colored boxes represent different motifs and their position in each BdMAPKK sequence.</p

Pairwise comparisons of the expression profiles of putative orthologs between rice and <i>B. distachyon</i>

<p><b>MAPKs and MAPKKs.</b> The mRNA relative amount represents the log 2 based value of expression intensity and the expression profiles of the two family genes in rice and <i>B. distachyon</i> were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046744#pone.0046744.s010" target="_blank">Table S6</a>. The treatment time (h) under the particular light and temperature condition is presented on the x-axis. R indicates the correlation coefficient of the expression profiles between orthologs or paralogs pairs under the corresponding light and temperature treatments. The positive (A), negative (B) and no obvious correlation (C) were detected from our identified 17 pairs of putative orthologs or paralogs in rice and <i>B. distachyon</i> MAPKs and MAPKKs.</p

Phylogenetic (A), EST (B) and RT-PCR (C) analyses of MAPKK genes.

<p>The gene names of each gene cluster originated from tandem or segmental duplication event were indicated with the same color (except the black color).</p