Covalent 14-3-3 Molecular Glues and Heterobifunctional Molecules Against Nuclear Transcription Factors and Regulators.

14-3-3 proteins have the unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that 14-3-3 interactions with specific phosphorylated substrate proteins can be enhanced through small-molecule natural product or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates as well as potential neo-substrates to sequester and inhibit their function has remained elusive. One of the 14-3-3 proteins, 14-3-3σ or SFN, has a cysteine C38 at the substrate binding interface near sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screened a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance interaction of 14-3-3σ with not only druggable transcription factors such as estrogen receptor (ERα), but also challenging oncogenic transcription factors such as YAP and TAZ that are part of the Hippo transducer pathway. We identified a hit EN171 that covalently targets 14-3-3 to enhance 14-3-3 interactions with ERα, YAP, and TAZ leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions, but also could be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and also demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

Antivirulence activities of retinoic acids against Staphylococcus aureus.

Multidrug-resistant bacteria such as Staphylococcus aureus constitute a global health problem. Gram-positive S. aureus secretes various toxins associated with its pathogenesis, and its biofilm formation plays an important role in antibiotic tolerance and virulence. Hence, we investigated if the metabolites of vitamin A1 might diminish S. aureus biofilm formation and toxin production. Of the three retinoic acids examined, 13-cis-retinoic acid at 10 µg/mL significantly decreased S. aureus biofilm formation without affecting its planktonic cell growth (MIC >400 µg/mL) and also inhibited biofilm formation by Staphylococcus epidermidis (MIC >400 µg/mL), but less affected biofilm formation by a uropathogenic Escherichia coli strain, a Vibrio strain, or a fungal Candida strain. Notably, 13-cis-retinoic acid and all-trans-retinoic acid significantly inhibited the hemolytic activity and staphyloxanthin production by S. aureus. Furthermore, transcriptional analysis disclosed that 13-cis-retinoic acid repressed the expressions of virulence- and biofilm-related genes, such as the two-component arlRS system, α-hemolysin hla, nuclease (nuc1 and nuc2), and psmα (phenol soluble modulins α) in S. aureus. In addition, plant and nematode toxicity assays showed that 13-cis-retinoic acid was only mildly toxic at concentrations many folds higher than its effective antibiofilm concentrations. These findings suggest that metabolites of vitamin A1, particularly 13-cis-retinoic acid, might be useful for suppressing biofilm formation and the virulence characteristics of S. aureus.

The Antibiofilm Effects of Antimony Tin Oxide Nanoparticles against Polymicrobial Biofilms of Uropathogenic Escherichia coli and Staphylococcus aureus.

Biofilms are responsible for persistent or recurring microbial infections. Polymicrobial biofilms are prevalent in environmental and medical niches. Dual-species biofilms formed by Gram-negative uropathogenic Escherichia coli (UPEC) and Gram-positive Staphylococcus aureus are commonly found in urinary tract infection sites. Metal oxide nanoparticles (NPs) are widely studied for their antimicrobial and antibiofilm properties. We hypothesized that antimony-doped tin (IV) oxide (ATO) NPs, which contain a combination of antimony (Sb) and tin (Sn) oxides, are good antimicrobial candidates due to their large surface area. Thus, we investigated the antibiofilm and antivirulence properties of ATO NPs against single- and dual-species biofilms formed by UPEC and S. aureus. ATO NPs at 1 mg/mL significantly inhibited biofilm formation by UPEC, S. aureus, and dual-species biofilms and reduced their main virulence attributes, such as the cell surface hydrophobicity of UPEC and hemolysis of S. aureus and dual-species biofilms. Gene expression studies showed ATO NPs downregulated the hla gene in S. aureus, which is essential for hemolysin production and biofilm formation. Furthermore, toxicity assays with seed germination and Caenorhabditis elegans models confirmed the non-toxic nature of ATO NPs. These results suggest that ATO nanoparticles and their composites could be used to control persistent UPEC and S. aureus infections.

Molecular detection and genotype analysis of Kudoa septempunctata from food poisoning outbreaks in Korea.

Concerns about foodborne illnesses caused by Kudoa septempunctata are steadily growing, but reports of K. septempunctata in clinical and food specimens related to food poisoning in Korea are limited. This study aimed to genetically identify K. septempunctata in patients with acute diarrhea and in clinical and food samples related to food poisoning caused by sashimi consumption. Both real-time and nested polymerase chain reaction assays were performed to detect K. septempunctata 18S and 28S rDNA genes in the stools of 348 patients with acute diarrhea, 11 samples (6 stool and 5 rectal swab samples) from patients with food poisoning, and 2 raw Paralichthys olivaceus samples collected from a restaurant where a food poisoning incident occurred. K. septempunctata was identified in 5 clinical specimens (4 stools and 1 rectal swab) and 1 P. olivaceus sashimi sample. All detected K. septempunctata were of genotype ST3. This is the first study to identify K. septempunctata in both patients and food samples with epidemiological relevance in Korea, providing evidence that it is a pathogen that causes food poisoning. Also, this is the first study to confirm the presence of K. septempunctata genes in rectal swabs. Despite continuing suspected occurrences of Kudoa foodborne outbreaks, the rate of identification of K. septempunctata is very low. One reason for this is the limitation in obtaining stool and vomit samples for the diagnosis of Kudoa infection. We strongly suggest the inclusion of rectal swabs among the diagnostic specimens for Kudoa food poisoning.

Peroxiredoxin5 Controls Vertebrate Ciliogenesis by Modulating Mitochondrial Reactive Oxygen Species.

AIMS: Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development. RESULTS: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells. INNOVATION: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS. CONCLUSION: Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

IFT46 plays crucial roles in craniofacial and cilia development.

The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development.

Perfluoroheptanoic acid affects amphibian embryogenesis by inducing the phosphorylation of ERK and JNK.

Perfluoroalkyl compounds (PFCs) are globally distributed synthetic compounds that are known to adversely affect human health. Developmental toxicity assessment of PFCs is important to facilitate the evaluation of their environmental impact. In the present study, we assessed the developmental toxicity and teratogenicity of PFCs with different numbers of carbon atoms on Xenopus embryogenesis. An initial frog embryo teratogenicity assay-Xenopus (FETAX) assay was performed that identified perfluorohexanoic (PFHxA) and perfluoroheptanoic (PFHpA) acids as potential teratogens and developmental toxicants. The mechanism underlying this teratogenicity was also investigated by measuring the expression of tissue-specific biomarkers such as phosphotyrosine­binding protein, xPTB (liver); NKX2.5 (heart); and Cyl18 (intestine). Whole­mount in situ hybridization, reverse transcriptase­polymerase chain reaction (RT-PCR), and histologic analyses detected severe defects in the liver and heart following exposure to PFHxA or PFHpA. In addition, immunoblotting revealed that PFHpA significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PFHxA slightly increased these, as compared with the control. These results suggest that PFHxA and PFHpA are developmental toxicants and teratogens, with PFHpA producing more severe effects on liver and heart development through the induction of ERK and JNK phosphorylation.

Characterization of ticlopidine-induced developmental and teratogenic defects in Xenopus embryos and human endothelial cells.

Ticlopidine is an anti-platelet drug that inhibits platelet aggregation via the functional alteration of platelet membranes. However, the mechanism underlying the adverse developmental effects of ticlopidine has not been clearly demonstrated. In this study, we evaluated the developmental toxicity and teratogenicity of ticlopidine on Xenopus laevis embryos and in human umbilical vein endothelial cells (HUVECs) using a frog embryo teratogenesis assay-Xenopus (FETAX) and blood and lymph vessel formation assays. Ticlopidine induced teratogenicity and inhibited growth, as evidenced by mortality rates and embryo lengths, respectively. Moreover, ticlopidine induced severe hemorrhages and inhibited both blood and lymph vessel formation by modulating the expression of xMsr and Prox1 in Xenopus embryos. Additionally, Nkx2.5 and Cyl104 levels were perturbed by ticlopidine exposure, and more extensive aberrations were observed in the liver and heart using whole-mount in situ hybridization. In addition, ticlopidine reduced branching in HUVECs by blocking the effect of the angiogenic vascular endothelial growth factor (VEGF). Results from this study suggest that ticlopidine is a developmental toxicant and teratogen and therefore this is a step forward in our understanding of the effects of ticlopidine during developmental processes.

EphB/ephrinB signaling in cell adhesion and migration.

Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.

Pick1 modulates ephrinB1-induced junctional disassembly through an association with ephrinB1.

Members of the Eph family have been implicated in the formation of cell-cell boundaries, cell movement, and positioning during development in the context of cancer progression. De-regulation of this signaling system is linked to the promotion of more aggressive and metastatic tumor phenotypes in a large variety of human cancers, including breast, lung, and prostate cancer, melanoma, and leukemia. Thus, it is interesting to consider the case of cancer progression where de-regulation of the Eph/ephrin signaling system results in invasion and metastasis. Here, we present evidence that Pick1, one of the essential components of the adherens junction, recovers ephrinB1-induced cell-cell de-adhesion. Loss of Pick1 leads to dissociation of epithelial cells via disruption of the adherens junction, a phenotype similar to ephrinB1 overexpression. In addition, overexpressed ephrinB1-induced disruption of the adherens junction is rescued via binding to Pick1. These data indicate that Pick1 is involved in regulating the cell-cell junction in epithelial cells, and this may influence therapeutic strategy decisions with regards to cell adhesion molecules in metastatic disease.

Characterization of atmospheric particles in Seoul, Korea using SEM-EDX.

Atmospheric particles in Seoul, Korea were investigated by scanning electron microscopy (SEM) equipped with energy dispersive X-ray analysis (EDX). Particles were identified and characterized by their morphology and elemental compositions. The morphology of particles was closely coupled with elemental compositions, which provided information on sources and transport processes of aerosols. There were various types of combustion-derived particles identified such as fly ashes, soot, organic matters, tar balls, chars, and sulfur-containing particles. These anthropogenic particles mainly have spherical shape with nano- to micro-meter size. Char was, however, distinguished by irregular shape with varying size up to several micrometers. The sulfur-containing aggregates show a wide range of size, shape, and elemental composition. In addition, bioaerosol and mineral dust were the most abundant particles from natural sources.