The Traitor

The proteolytic activation of protein kinase Cδ (PKCδ) generates a catalytic fragment called PKCδ-CF, which induces cell death. However, the mechanisms underlying PKCδ-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKCδ-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKCδ-CF expression. Totally, 3000 phosphorylation sites were analyzed. Considering the fact that early responses to PKCδ-CF expression initiate cell death, we sought to identify pathways possibly related directly with PKCδ by further analyzing the data set of phosphorylation events that occur in the initiation stage of cell death. Interacting analysis of this data set indicates that PKCδ-CF triggers complicated networks to initiate cell death, and motif analysis and biochemistry verification reveal that several kinases in the downstream of PKCδ conduct these networks. By analysis of the specific sequence motif of kinase-substrate, we also find 59 candidate substrates of PKCδ from the up-regulated phosphopeptides, of which 12 were randomly selected for <i>in vitro</i> kinase assay and 9 were consequently verified as substrates of PKCδ. To our greatest understanding, this study provides the most systematic analysis of phosphorylation events initiated by the cleaved activated PKCδ, which would vastly extend the profound understanding of PKCδ-directed signal pathways in cell death. The MS data have been deposited to the ProteomeXchange with identifier PXD000225

Structure and Absolute Configuration of Jurassic Polyketide-Derived Spiroborate Pigments Obtained from Microgram Quantities

Complete structural elucidation of natural products is often challenging due to structural complexity and limited availability. This is true for present-day secondary metabolites, but even more for exceptionally preserved secondary metabolites of ancient organisms that potentially provide insights into the evolutionary history of natural products. Here, we report the full structure and absolute configuration of the borolithochromes, enigmatic boron-containing pigments from a Jurassic putative red alga, from samples of less than 50 μg using microcryo­probe NMR, circular dichroism spectroscopy, and density functional theory calculations and reveal their polyketide origin. The pigments are identified as spiroborates with two pentacyclic <i>sec</i>-butyl-trihydroxy-methyl-benzo­[<i>gh</i>]­tetraphen-one ligands and less-substituted derivatives. The configuration of the <i>sec</i>-butyl group is found to be (<i>S</i>). Because the exceptional benzo­[<i>gh</i>]­tetraphene scaffold is otherwise only observed in the recently discovered polyketide clostrubin from a present-day <i>Clostridium</i> bacterium, the Jurassic borolithochromes now can be unambiguously linked to the modern polyketide, providing evidence that the fossil pigments are almost originally preserved secondary metabolites and suggesting that the pigments in fact may have been produced by an ancient bacterium. The borolithochromes differ fundamentally from previously described boronated polyketides and represent the first boronated aromatic polyketides found so far. Our results demonstrate the potential of microcryoprobe NMR in the analysis of previously little-explored secondary metabolites from ancient organisms and reveal the evolutionary significance of clostrubin-type polyketides

Phosphonium-Substituted Conjugated Polyelectrolytes Display Efficient Visible-Light-Induced Antibacterial Activity

We report the light-activated antibacterial activity of a new class of phosphonium (R-PMe3+)-substituted conjugated polyelectrolytes (CPEs). These polyelectrolytes feature a poly(phenylene ethynylene) (PPE) conjugated backbone substituted with side groups with the structure −O–(CH2)nPMe3+, where n = 3 or 6. The length of the side groups has an effect on the hydrophobic character of the CPEs and their propensity to interact with bacterial membranes. In a separate study, these phosphonium-substituted PPE CPEs were demonstrated to photosensitize singlet oxygen (1O2) and reactive oxygen species, a key factor for the photoinduced inactivation of bacteria. In this study, in vitro antibacterial assays against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were performed by employing the series of polyelectrolytes under both dark and illumination conditions. In general, the phosphonium-substituted CPEs displayed profound light-activated biocidal activity, with >99% colony forming unit (CFU) reduction after 15 min of light exposure (16 mW cm–2) at a ≤20 μM CPE concentration. Strong biocidal activity was also observed in the dark for a CPE concentration of 20 μM against S. aureus; however, higher concentrations (200 μM) were needed to enable dark inactivation of E. coli. The dark activity is ascribed to bacterial membrane disruption by the CPEs, supported by a correlation of dark biocidal activity with the chain length of the side groups. The light-activated biocidal activity is associated with the ability of the CPEs to sensitize ROS, which is cytotoxic to the microorganisms. Serial dilution bacterial plating experiments revealed that the series of CPEs was able to induce a >5-log kill versus E. coli with 15 min of exposure to a blue LED source (16 mW cm–2)

A Novel Semantics-Preserving Hashing for Fine-Grained Image Retrieval

With the advent of the era of big data, the storage and retrieval of data have become a research hotspot. Hashing methods that transform high-dimensional data into compact binary codes have received increasing attention. Recently, with the successful application of convolutional neural networks in computer vision, deep hashing methods utilize an end-to-end framework to learn feature representations and hash codes mutually, which achieve better retrieval performance than conventional hashing methods. However, deep hashing methods still face some challenges in image retrieval. Firstly, most existing deep hashing methods preserve similarity between original data space and hash coding space using loss functions with high time complexity, which cannot get a win-win situation in time and accuracy. Secondly, few existing deep hashing methods are designed for fine-grained image retrieval, which is necessary in practice. In this study, we propose a novel semantics-preserving hashing method which solves the above problems. We add a hash layer before the classification layer as a feature switch layer to guide the classification. At the same time, we replace the complicated loss with the simple classification loss, combining with quantization loss and bit balance loss to generate high-quality hash codes. Besides, we incorporate feature extractor designed for fine-grained image classification into our network for better representation learning. The results on three widely-used fine-grained image datasets show that our method is superior to other state-of-the-art image retrieval methods

Synthesis of a Novel Quinoline Skeleton Introduced Cationic Polyfluorene Derivative for Multimodal Antimicrobial Application

A new functional polyfluorene derivative containing quinoline skeleton and quarternary ammonium group (QAG) modified side chains (PFPQ) was synthesized and characterized. The multimodal antimicrobial effect toward Gram-negative E. coli was achieved by the dark toxicity resulting from the quinoline skeleton, QAG, and light toxicity resulting from reactive oxygen species (ROS) produced by the main backbone of PFPQ under white light. The mechanism of interaction between PFPQ and bacteria was also demonstrated. PFPQ bound to E. coli mainly through electrostatic interactions causing nearly 50% bacterial death in the absence of light irradiation, and the huge capability of PFPQ to generate ROS under white light opened another bactericidal mode. The killing efficiency was more than 99% upon relatively mild irradiation under white light (400–800 nm) with a light dose of 18 J·cm^–2. PFPQ with the incorporation of quinoline into the backbones will provide a new versatile strategy to achieve the multimodal antimicrobial effect to fight against resistant bacteria

Boosting Type‑I and Type-II ROS Production of Water-Soluble Porphyrin for Efficient Hypoxic Tumor Therapy

As the most successful clinically approved photosensitizers, porphyrins have been extensively employed in the photodynamic therapy (PDT) of cancers. However, their poor water solubility, aggregation-induced self-quenching on ROS generation, and a low tolerance for a hypoxic condition usually result in unsatisfied therapeutic outcomes. Therefore, great efforts have been dedicated to improving the PDT efficacy of porphyrin-type photosensitizers in treating hypoxic tumors, including combination with additional active components or therapies, which can significantly complicate the therapeutic process. Herein, we report a novel water-soluble porphyrin with O-linked cationic side chains, which exhibits good water solubility, high photostability, and significantly enhanced ROS generation efficacy in both type-I and type-II photodynamic pathways. We have also found that the end charges of side chains can dramatically affect the ROS generation of the porphyrin. The cationic porphyrin exhibited high in vitro PDT efficacy with low IC50 values both in normoxia and hypoxia. Hence, during in vivo PDT study, the cationic porphyrin displayed highly effective tumor ablation capability. This study demonstrates the power of side-chain chemistry in tuning the photodynamic property of porphyrin, which offers a new effective strategy to enhance the anticancer performance of photosensitizers for fulfilling the increasing demands for cancer therapy in clinics

Regulation of Active DNA Demethylation by a Methyl-CpG-Binding Domain Protein in <i>Arabidopsis thaliana</i>

<div><p>Active DNA demethylation plays crucial roles in the regulation of gene expression in both plants and animals. In <i>Arabidopsis thaliana</i>, active DNA demethylation is initiated by the ROS1 subfamily of 5-methylcytosine-specific DNA glycosylases via a base excision repair mechanism. Recently, IDM1 and IDM2 were shown to be required for the recruitment of ROS1 to some of its target loci. However, the mechanism(s) by which IDM1 is targeted to specific genomic loci remains to be determined. Affinity purification of IDM1- and IDM2- associating proteins demonstrated that IDM1 and IDM2 copurify together with two novel components, methyl-CpG-binding domain protein 7 (MBD7) and IDM2-like protein 1 (IDL1). IDL1 encodes an α-crystallin domain protein that shows high sequence similarity with IDM2. MBD7 interacts with IDM2 and IDL1 <i>in vitro</i> and <i>in vivo</i> and they form a protein complex associating with IDM1 <i>in vivo</i>. MBD7 directly binds to the target loci and is required for the H3K18 and H3K23 acetylation <i>in planta</i>. <i>MBD7</i> dysfunction causes DNA hypermethylation and silencing of reporter genes and a subset of endogenous genes. Our results suggest that a histone acetyltransferase complex functions in active DNA demethylation and in suppression of gene silencing at some loci in <i>Arabidopsis</i>.</p></div

Protein-protein interactions between IDM1, IDM2, IDL1 and MBD7.

<p>(A) Yeast two-hybrid assays. Yeast cells carrying different fusion protein combinations are listed on the left. Yeast cells expressing the indicated proteins from the pGBK-T7 (BD) and pGAD-T7 (AD) vectors were plated onto medium lacking Leu and Trp (SD-LT) (left) or medium lacking Leu, Trp, Ade and His (SD-LTHA) (right). <b>(B)</b> Pull-down assays showing that IDM2, IDL1 and MBD7 interact with each other. <b>(C)</b> Split-luc assays showing that IDL1 and MBD7 can interact with IDM2 in <i>N</i>. <i>benthamiana</i> leaves. Three biological replicates were performed, and similar results were obtained. (D) Co-immunoprecipitation of MBD7 with IDM2 or IDL1 in tobacco leaves. MYC-tagged IDM2 and GFP-tagged IDM1 were transiently expressed in <i>N</i>. <i>benthamiana</i> leaves. Anti-GFP was used for immunoprecipitation (IP); anti-MYC and anti-GFP were used for immunoblotting; Input, total protein before immunoprecipitation. Transgenic plants expressing MBD7-Myc or IDM1-HA-YFP under their native promoters and their F1 offspring were used for co-IP.</p

Working model for the IDM1-IDM2-IDL1-MBD7 complex functioning in ROS1 mediated active DNA demethylation at some loci in <i>Arabidopsis</i>.

<p>MBD7 forms a complex with IDM1, IDM2 and IDL1, and recognizes methylated DNA through methyl-CpG-binding domains. Then IDM1 is recruited to specific loci and acetylates histone H3 at K18 and K23, facilitating active DNA demethylation by ROS1.</p

Histone acetylation marks and MBD7 association with chromatin.

<p>(A) H3K18 acetylation (ac) and H3K23ac levels at the hyper-DMR loci and control regions. ChIP was performed with antibodies against H3K18ac and H3K23ac. The ChIP signal was quantified relative to input DNA. The no-antibody precipitates served as negative control. Two biological replicates were performed, and very similar results were obtained. Standard errors were calculated from three technical repeats, *P < 0.05. (B) Association of MBD7 protein with hyper-DMR loci. ChIP was performed in WT and <i>MBD7-Myc</i> transgenic plants with antibody against Myc.</p