45 research outputs found
The Traitor
The
proteolytic activation of protein kinase CĪ“ (PKCĪ“)
generates a catalytic fragment called PKCĪ“-CF, which induces
cell death. However, the mechanisms underlying PKCĪ“-CF-mediated
cell death are largely unknown. On the basis of an engineering leukemic
cell line with inducible expression of PKCĪ“-CF, here we employ
SILAC-based quantitative phosphoproteomics to systematically and dynamically
investigate the overall phosphorylation events during cell death triggered
by PKCĪ“-CF expression. Totally, 3000 phosphorylation sites were
analyzed. Considering the fact that early responses to PKCĪ“-CF
expression initiate cell death, we sought to identify pathways possibly
related directly with PKCĪ“ 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 PKCĪ“-CF
triggers complicated networks to initiate cell death, and motif analysis
and biochemistry verification reveal that several kinases in the downstream
of PKCĪ“ conduct these networks. By analysis of the specific
sequence motif of kinase-substrate, we also find 59 candidate substrates
of PKCĪ“ 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 PKCĪ“. To our greatest
understanding, this study provides the most systematic analysis of
phosphorylation events initiated by the cleaved activated PKCĪ“,
which would vastly extend the profound understanding of PKCĪ“-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 Ī¼g using microcryoĀprobe
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-benzoĀ[<i>gh</i>]Ātetraphen-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 benzoĀ[<i>gh</i>]Ātetraphene 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 āOā(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 cmā2) at a ā¤20 Ī¼M CPE concentration. Strong
biocidal activity was also observed in the dark for a CPE concentration
of 20 Ī¼M against S. aureus; however, higher
concentrations (200 Ī¼M) 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 cmā2)
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 (400ā800 nm) with a light dose
of 18 JĀ·cm<sup>ā2</sup>. 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 TypeāI 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 Ī±-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