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Find video protocols related to scientific articles indexed in Pubmed.
Co-intercalation of Acid Red 337 and an UV Absorbent into Layered Double Hydroxides: Enhancement of Photostability.
ACS Appl Mater Interfaces
PUBLISHED: 11-19-2014
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Organic-inorganic hybrid pigments with enhanced thermo- and photostability have been prepared by co-intercalating C.I. Acid Red 337 (AR337) and an UV absorbent (BP-4) into the interlayer of ZnAl layered double hydroxides through a co-precipitation method. The obtained compounds were characterized by XRD, FT-IR, SEM, TG-DTG-DTA, UV-vis and CIE 1976 L*a*b* color scales. The results show the successful co-intercalation of AR337 and BP-4 into the interlayer region of LDHs and reveal the presence of host-guest interactions between LDHs host layers and guest anions of AR337 and BP-4 as well as guest-guest interactions between AR337 and BP-4. The intercalation can improve the thermostability of AR337 due to the protection of LDHs layers. Moreover, the co-intercalation of AR337 and BP-4 not only markedly enhances the photostability of AR337 but also has significant influence on the color property of the hybrid pigment.
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Synthesis, structure-activity relationship studies, and antibacterial evaluation of 4-chromanones and chalcones, as well as olympicin a and derivatives.
J. Med. Chem.
PUBLISHED: 10-07-2014
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On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 ?g/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2',4'-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.
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[Effects of microRNA-294 on inflammatory factor of sepsis by targeting triggering receptor expressed on myeloid cells-1].
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue
PUBLISHED: 09-19-2014
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To investigate the effects of microRNA-294 (miR-294) on tumor necrosis factor-? (TNF-?), interleukin -6 (IL-6) and high mobility group box 1 (HMGB1) secretion in sepsis by targeting triggering receptor expressed on myeloid cell-1 (TREM-1).
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Progress in the discovery of treatments for C. difficile infection: A clinical and medicinal chemistry review.
Curr Top Med Chem
PUBLISHED: 08-26-2014
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Clostridium difficile is an anaerobic, Gram-positive pathogen that causes C. difficile infection, which results in significant morbidity and mortality. The incidence of C. difficile infection in developed countries has become increasingly high due to the emergence of newer epidemic strains, a growing elderly population, extensive use of broad spectrum antibiotics, and limited therapies for this diarrheal disease. Because treatment options currently available for C. difficile infection have some drawbacks, including cost, promotion of resistance, and selectivity problems, new agents are urgently needed to address these challenges. This review article focuses on two parts: the first part summarizes current clinical treatment strategies and agents under clinical development for C. difficile infection; the second part reviews newly reported anti-difficile agents that have been evaluated or reevaluated in the last five years and are in the early stages of drug discovery and development. Antibiotics are divided into natural product inspired and synthetic small molecule compounds that may have the potential to be more efficacious than currently approved treatments. This includes potency, selectivity, reduced cytotoxicity, and novel modes of action to prevent resistance.
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Hepatocyte growth factor (Hgf) stimulates low density lipoprotein receptor-related protein (Lrp) 5/6 phosphorylation and promotes canonical Wnt signaling.
J. Biol. Chem.
PUBLISHED: 04-01-2014
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While Wnt and Hgf signaling pathways are known to regulate epithelial cell responses during injury and repair, whether they exhibit functional cross-talk is not well defined. Canonical Wnt signaling is initiated by the phosphorylation of the Lrp5/6 co-receptors. In the current study we demonstrate that Hgf stimulates Met and Gsk3-dependent and Wnt-independent phosphorylation of Lrp5/6 at three separate activation motifs in subconfluent, de-differentiated renal epithelial cells. Hgf treatment stimulates the selective association of active Gsk3 with Lrp5/6. In contrast, Akt-phosphorylated inactive Gsk3 is excluded from this association. Hgf stimulates ?-catenin stabilization and nuclear accumulation and protects against epithelial cell apoptosis in an Lrp5/6-dependent fashion. In vivo, the increase in Lrp5/6 phosphorylation and ?-catenin stabilization in the first 6-24 h after renal ischemic injury was significantly reduced in mice lacking Met receptor in the renal proximal tubule. Our results thus identify Hgf as an important transactivator of canonical Wnt signaling that is mediated by Met-stimulated, Gsk3-dependent Lrp5/6 phosphorylation.
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SRF is required for neutrophil migration in response to inflammation.
Blood
PUBLISHED: 02-26-2014
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Serum response factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of SRF loss in neutrophils, we crossed Srf(fl/fl) mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in Srf knockout (KO) neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to N-formyl-methionine-leucine-phenylalanine, resulting in significantly disrupted cytoskeletal remodeling. Srf KO neutrophils fail to migrate to sites of inflammation in vivo and along chemokine gradients in vitro. Polarization in response to cytokine stimuli is absent and Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in cellular adhesion, and although integrin expression levels are maintained with loss of SRF, integrin activation and trafficking are disrupted. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, underscoring an essential function for SRF and its pathway in health and disease.
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Herbicidin congeners, undecose nucleosides from an organic extract of Streptomyces sp. L-9-10.
J. Nat. Prod.
PUBLISHED: 02-17-2014
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Four new undecose nucleosides (herbicidin congeners), three known herbicidins, and 9-(?-d-arabinofuranosyl)hypoxanthine (Ara-H) were isolated from the organic extract of a fermentation culture of Streptomyces sp. L-9-10 using proton NMR-guided fractionation. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry analyses. These structures included 2'-O-demethylherbicidin F (1), 9'-deoxy-8',8'-dihydroxyherbicidin B (2), 9'-deoxy-8'-oxoherbicidin B (2a), and the 8'-epimer of herbicidin B (3). This is the first detailed assignment of proton and carbon chemical shifts for herbicidins A, B, and F. The isolated compounds were evaluated for cancer chemopreventive potential based on inhibition of tumor necrosis factor alpha (TNF-?)-induced nuclear factor-kappa B (NF-?B) activity.
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Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux.
Nat. Med.
PUBLISHED: 01-26-2014
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Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.
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Synthesis and antibacterial evaluation of anziaic acid and analogues as topoisomerase I inhibitors.
Medchemcomm
PUBLISHED: 12-24-2013
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Naturally occurring anziaic acid was very recently reported as a topoisomerase I inhibitor with antibacterial activity. Herein total synthesis of anziaic acid and structural analogues is described and the preliminary structure-activity relationship (SAR) has been developed based on topoisomerase inhibition and whole cell antibacterial activity.
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Inhibitory Effect of a Callophycin A Derivative on iNOS Expression via Inhibition of Akt in Lipopolysaccharide-Stimulated RAW 264.7 Cells.
J. Nat. Prod.
PUBLISHED: 12-03-2013
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In previous studies, (R)-2-isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2,3(9H)-dicarboxylate (1), a callophycin A derivative, was found to strongly inhibit nitrite production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, while (R)- or (S)-callophycin A showed only weak inhibition. We currently report additional studies to define the mechanisms underlying the inhibitory action of 1. Expression of inducible nitric oxide synthase (iNOS) was reduced at both protein and mRNA levels. Major upstream signaling molecules and transcription factors regulating iNOS expression were examined, but it was found that 1 did not affect the phosphorylated and total protein levels of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and signal transducer and activator of transcription 1 (STAT1), nor did it mediate the degradation of the inhibitor of nuclear factor-?B ?-isoform (I?B?). However, starting at early time points, 1 consistently inhibited the phosphorylation of protein kinase B/Akt at serine 473. In addition, 1 suppressed the protein expression of octamer-binding transcription factor-2 (Oct-2) and the expression of microRNA 155 (miR-155). In sum, compound 1 inhibits LPS-induced nitrite production by a unique and complex mechanism. Reduction of iNOS expression is accompanied by inhibition of Akt activation, Oct-2 protein expression, and miR-155 expression.
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Different Raf Protein Kinases Mediate Different Signaling Pathways to Stimulate E3 Ligase RFFL Gene Expression in Cell Migration Regulation.
J. Biol. Chem.
PUBLISHED: 10-10-2013
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We previously characterized a G?12-specific signaling pathway that stimulates the transcription of the E3 ligase RFFL via the protein kinase ARAF and ERK. This pathway leads to persistent PKC activation and is important for sustaining fibroblast migration. However, questions remain regarding how G?12 specifically activates ARAF, which transcription factor is involved in G?12-mediated RFFL expression, and whether RFFL is important for cell migration stimulated by other signaling mechanisms that can activate ERK. In this study, we show that replacement of the G?12 residue Arg-264 with Gln, which is the corresponding G?13 residue, abrogates the ability of G?12 to interact with or activate ARAF. We also show that G?12 can no longer interact with and activate an ARAF mutant with its C-terminal sequence downstream of the kinase domain being replaced with the corresponding CRAF sequence. These results explain why G?12, but not G?13, specifically activates ARAF but not CRAF. Together with our finding that recombinant G?12 is sufficient for stimulating the kinase activity of ARAF, this study reveals an ARAF activation mechanism that is different from that of CRAF. In addition, we show that this G?12-ARAF-ERK pathway stimulates RFFL transcription through the transcription factor c-Myc. We further demonstrate that EGF, which signals through CRAF, and an activated BRAF mutant also activate PKC and stimulate cell migration through up-regulating RFFL expression. Thus, RFFL-mediated PKC activation has a broad significance in cell migration regulation.
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Au-Pd nanoalloys supported on Mg-Al mixed metal oxides as a multifunctional catalyst for solvent-free oxidation of benzyl alcohol.
Dalton Trans
PUBLISHED: 08-24-2013
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Au-Pd nanoalloys supported on Mg-Al mixed metal oxides prepared using sol-immobilisation are found to be highly efficient and reusable catalysts for the solvent-free oxidation of benzyl alcohol using molecular oxygen under low pressure. When using this support alloying Pd with Au resulted in an increase in both activity and selectivity to benzaldehyde and moreover an improved resistance to catalyst deactivation compared with the monometallic Pd and Au catalysts. The turnover number for the Au/Pd 1:1 molar ratio catalyst achieved 13,000 after 240 min and the selectivity to benzaldehyde was maintained at 93%; this high catalytic activity can be retained in full after three successive uses. The ensemble and electronic effect of Au-Pd nanoalloys were studied by IR spectroscopy using CO chemisorption, XPS and HRTEM. Moreover, the bifunctional nature of the acid-base MgAl-MMO support was found to be important as the acid sites are considered to be responsible for the improvement of catalytic activity; while, the basic sites gave rise to high selectivity. A possible mechanism with Au-Pd nanoparticles as the active sites has been proposed, illustrating that the oxidation of benzyl alcohol can proceed through the cooperation between the Au-Pd nanoalloys and the base/acid sites on the surface of the support.
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Synthesis of quantum-sized ZnO nanoparticles in different medium and their application to NO2 sensors.
J Nanosci Nanotechnol
PUBLISHED: 05-08-2013
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Quantum-sized ZnO nanoparticles were synthesized using zinc acetate dihydrate through a sol-gel process in different mediums: water, ethanol and methanol. Three types of modifiers: tetraethyl orthosilicate (TEOS), sodium dodecyl sulfate (SDS) and oleic acid (OA) were added to control the growth of the ZnO nanoparticles and inhibit Ostwald ripening. X-ray Diffraction (XRD) analyses revealed that ZnO have a hexagonal crystal structure, the estimated average crystallite sizes of modified ZnO are in the range of 4.5-10 nm, while the crystallite sizes of non-modified ZnO are large than 20 nm. X-ray photoelectron spectroscopy (XPS) analyses obtained the surface composition and chemical states of the products of ZnO. In this paper, the obtained quantum-sized ZnO nanoparticles as a novel sensing material were used to detect NO2 in environment. The sensing tests indicated that the ZnO based sensors not only have high response to NO2 but also exhibited high selectivity to CO and CH4 at low operating temperature of 290 degrees C.
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A network of interactions enables CCM3 and STK24 to coordinate UNC13D-driven vesicle exocytosis in neutrophils.
Dev. Cell
PUBLISHED: 05-06-2013
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Neutrophil degranulation plays an important role in acute innate immune responses and is tightly regulated because the granule contents can cause tissue damage. However, this regulation remains poorly understood. Here, we identify the complex of STK24 and CCM3 as being an important regulator of neutrophil degranulation. Lack of either STK24 or CCM3 increases the release of a specific granule pool without affecting other neutrophil functions. STK24 appears to suppress exocytosis by interacting and competing with UNC13D C2B domain for lipid binding, whereas CCM3 has dual roles in exocytosis regulation. Although CCM3 stabilizes STK24, it counteracts STK24-mediated inhibition of exocytosis by recruiting STK24 away from the C2B domain through its Ca(2+)-sensitive interaction with UNC13D C2A domain. This STK24/CCM3-regulated exocytosis plays an important role in the protection of kidneys from ischemia-reperfusion injury. Together, these findings reveal a function of the STK24 and CCM3 complex in the regulation of ligand-stimulated exocytosis.
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Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-09-2013
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Increasing evidence points to a role for the protein quality control in the endoplasmic reticulum (ER) in maintaining intestinal homeostasis. However, the specific role for general ER chaperones in this process remains unknown. Herein, we report that a major ER heat shock protein grp94 interacts with MesD, a critical chaperone for the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to the cell surface, resulting in a profound loss of canonical Wnt signaling. The significance of this finding is demonstrated in vivo in that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear ?-catenin translocation, loss of crypt-villus structure, and impaired barrier function. Taken together, our work has uncovered the role of grp94 in chaperoning LRP6-MesD in coordinating intestinal homeostasis, placing canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER.
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Macrocyclic drugs and synthetic methodologies toward macrocycles.
Molecules
PUBLISHED: 04-03-2013
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Macrocyclic scaffolds are commonly found in bioactive natural products and pharmaceutical molecules. So far, a large number of macrocyclic natural products have been isolated and synthesized. The construction of macrocycles is generally considered as a crucial and challenging step in the synthesis of macrocyclic natural products. Over the last several decades, numerous efforts have been undertaken toward the synthesis of complex naturally occurring macrocycles and great progresses have been made to advance the field of total synthesis. The commonly used synthetic methodologies toward macrocyclization include macrolactonization, macrolactamization, transition metal-catalyzed cross coupling, ring-closing metathesis, and click reaction, among others. Selected recent examples of macrocyclic synthesis of natural products and druglike macrocycles with significant biological relevance are highlighted in each class. The primary goal of this review is to summarize currently used macrocyclic drugs, highlight the therapeutic potential of this underexplored drug class and outline the general synthetic methodologies for the synthesis of macrocycles.
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Syntheses and evaluation of macrocyclic engelhardione analogs as antitubercular and antibacterial agents.
J. Antibiot.
PUBLISHED: 04-03-2013
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The natural product engelhardione is an underexplored chemotype for developing novel treatments for bacterial infections; we therefore explored this natural product scaffold for chemical diversification and structure-activity relationship studies. Macrocyclic engelhardione and structural regioisomers were synthesized using a series of aldol condensations and selective hydrogenations to generate the 1,7-diarylheptan-3-one derivatives, followed by microwave-assisted intramolecular Ullmann coupling to afford a series of macrocyclic diaryl ether analogs. An extended macrocyclic chemical library was then produced by oxime formation, reductive amination and O-alkylation. Antibacterial evaluation revealed that the reductive amination derivatives 7b and 7d showed moderate activities (minimum inhibitory concentrations: 12.5-25??g?ml(-1)) against Mycobacterium tuberculosis and Gram-positive pathogens, as well as anti-Gram-negative activity against an efflux impaired Escherichia coli strain. These results provide validated leads for further optimization and development.
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Genetic deletion of catalytic subunits of AMP-activated protein kinase increases osteoclasts and reduces bone mass in young adult mice.
J. Biol. Chem.
PUBLISHED: 03-13-2013
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AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis and a potential therapeutic target for the intervention of cancer and metabolic disorders. However, the role of AMPK in bone homeostasis remains incompletely understood. Here we assessed the skeletal phenotype of mice lacking catalytic subunits of AMPK and found that mice lacking AMPK?1 (Prkaa1(-/-)) or AMPK?2 (Prkaa2(-/-)) had reduced bone mass compared with the WT mice, although the reduction was less in Prkaa2(-/-) mice than in Prkaa1(-/-) mice. Static and dynamic bone histomorphometric analyses revealed that Prkaa1(-/-) mice had an elevated rate of bone remodeling because of increases in bone formation and resorption, whereas AMPK?2 KO-induced bone mass reduction was largely attributable to elevated bone resorption. In agreement with our in vivo results, AMPK? deficiency was associated with increased osteoclastogenesis in vitro. Moreover, we found that AMPK?1 inhibited the receptor activator of nuclear factor ?B (RANK) signaling, providing an explanation for AMPK-mediated inhibition of osteoclastogenesis. Therefore, our findings further underscore the importance of AMPK in bone homeostasis, in particular osteoclastogenesis, in young adult mammals.
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Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes.
J. Cell Biol.
PUBLISHED: 02-11-2013
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Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P(2)) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P(2) regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P(2), and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P(2) hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P(2) promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to ?-catenin stabilization.
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Role for the guanine nucleotide exchange factor phosphatidylinositol-3,4,5-trisphosphate-dependent rac exchanger 1 in platelet secretion and aggregation.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 12-29-2011
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Recent studies have shown a role for Rac1 in regulating platelet functions, but how Rac1 is activated in platelets remains unclear. Phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) was originally identified in neutrophils that regulates phagocyte functions. We sought to examine whether P-Rex1 plays a role in platelet activation.
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A PLC?/PI3K?-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis.
Dev. Cell
PUBLISHED: 10-20-2011
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Neutrophils, in response to a chemoattractant gradient, undergo dynamic F-actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLC?-PKC and PI3K?-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.
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Total synthesis and structural revision of engelhardione.
Tetrahedron Lett.
PUBLISHED: 09-20-2011
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The total synthesis of the macrocyclic natural product engelhardione is reported. This effort led to the structural revision of the published structure of engelhardione to that of pterocarine. The revision reflects the change of the substitution pattern of one phenyl ether ring from the meta to the para position. To confirm, pterocarine (2) and its close regioisomer 3 were subsequently synthesized for comparison. Moreover, to the best of our knowledge, our synthesis of 1 represents the first example of a 14-membered macrocyclic diarylheptanoid with a meta-meta substitution pattern at the diphenyl ether moiety.
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Design, synthesis, and biological evaluation of callophycin A and analogues as potential chemopreventive and anticancer agents.
Bioorg. Med. Chem.
PUBLISHED: 07-16-2011
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Callophycin A was originally isolated from the red algae Callophycus oppositifolius and shown to mediate anticancer and cytotoxic effects. In our collaborative effort to identify potential chemopreventive and anticancer agents with enhanced potency and selectivity, we employed a tetrahydro-?-carboline-based template inspired by callophycin A for production of a chemical library. Utilizing a parallel synthetic approach, 50 various functionalized tetrahydro-?-carboline derivatives were prepared and assessed for activities related to cancer chemoprevention and cancer treatment: induction of quinone reductase 1 (QR1) and inhibition of aromatase, nitric oxide (NO) production, tumor necrosis factor (TNF)-?-induced NF?B activity, and MCF7 breast cancer cell proliferation. Biological results showed that the n-pentyl urea S-isomer 6a was the strongest inducer of QR1 with an induction ratio (IR) value of 4.9 at 50 ?M [the concentration to double the activity (CD)=3.8 ?M] and its corresponding R-isomer 6f had an IR value of 4.3 (CD=0.2 ?M). The isobutyl carbamate derivative 3d with R stereochemistry demonstrated the most potent inhibitory activity of NF?B, with the half maximal inhibitory concentration (IC(50)) value of 4.8 ?M, and also showed over 60% inhibition at 50 ?M of NO production (IC(50)=2.8 ?M). The R-isomer urea derivative 6j, having an appended adamantyl group, exhibited the most potent MCF7 cell proliferation inhibitory activity (IC(50)=14.7 ?M). The S-isomer 12a of callophycin A showed the most potent activity in aromatase inhibition (IC(50)=10.5 ?M).
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The structure-activity relationship of urea derivatives as anti-tuberculosis agents.
Bioorg. Med. Chem.
PUBLISHED: 06-12-2011
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The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure-activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.
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PI3K? deletion reduces variability in the in vivo osteolytic response induced by orthopaedic wear particles.
J. Orthop. Res.
PUBLISHED: 03-31-2011
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Orthopedic wear particles activate a number of intracellular signaling pathways associated with inflammation in macrophages and we have previously shown that the phosphoinositol-3-kinase (PI3K)/Akt pathway is one of the signal transduction pathways that mediates the in vitro activation of macrophages by orthopedic wear particles. Since PI3K? is primarily responsible for PI3K activity during inflammation, we hypothesized that PI3K? mediates particle-induced osteolysis in vivo. Our results do not strongly support the hypothesis that PI3K? regulates the overall amount of particle-induced osteolysis in the murine calvarial model. However, our results strongly support the conclusion that variability in the amount of particle-induced osteolysis between individual mice is reduced in the PI3K?(-/-) mice. These results suggest that PI3K? contributes to osteolysis to different degrees in individual mice and that the mice, and patients, that are most susceptible to osteolysis may be so, in part, due to an increased contribution from PI3K?.
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Evidence for direct activation of mTORC2 kinase activity by phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem.
PUBLISHED: 02-10-2011
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mTORC2 (mammalian target of rapamycin complex 2) plays important roles in signal transduction by regulating an array of downstream effectors, including protein kinase AKT. However, its regulation by upstream regulators remains poorly characterized. Although phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is known to regulate the phosphorylation of AKT Ser(473), the hydrophobic motif (HM) site, by mTORC2, it is not clear whether PtdIns(3,4,5)P(3) can directly regulate mTORC2 kinase activity. Here, we used two membrane-docked AKT mutant proteins, one with and the other without the pleckstrin homology (PH) domain, as substrates for mTORC2 to dissect the roles of PtdIns(3,4,5)P(3) in AKT HM phosphorylation in cultured cells and in vitro kinase assays. In HEK293T cells, insulin and constitutively active mutants of small GTPase H-Ras and PI3K could induce HM phosphorylation of both AKT mutants, which was blocked by the PI3K inhibitor LY294002. Importantly, PtdIns(3,4,5)P(3) was able to stimulate the phosphorylation of both AKT mutants by immunoprecipitated mTOR2 complexes in an in vitro kinase assay. In both in vivo and in vitro assays, the AKT mutant containing the PH domain appeared to be a better substrate than the one without the PH domain. Therefore, these results suggest that PtdIns(3,4,5)P(3) can regulate HM phosphorylation by mTORC2 via multiple mechanisms. One of the mechanisms is to directly stimulate the kinase activity of mTORC2.
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Phospholipase C-?3 regulates Fc?RI-mediated mast cell activation by recruiting the protein phosphatase SHP-1.
Immunity
PUBLISHED: 01-18-2011
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Mast cells are major effectors in high-affinity IgE receptor (Fc?RI)-dependent allergic reactions. Here we show that phospholipase C (PLC)-?3 is crucial for Fc?RI-mediated mast cell activation. Plcb3(-/-) mice showed blunted Fc?RI-dependent late-phase, but not acute, anaphylactic responses and airway inflammation. Accordingly, Fc?RI stimulation of Plcb3(-/-) mast cells exhibited reduced cytokine production but normal degranulation. Reduced cytokine production in Plcb3(-/-) cells could be accounted for by increased activity of the negative regulatory Src family kinase Lyn and reduced activities of the positive regulatory protein kinases MAPKs. Mechanistically, PLC-?3 constitutively interacts with Fc?RI, Lyn, and SHP-1 (protein phosphatase). SHP-1 probably recognizes its substrates Lyn and MAPKs via the recently described kinase tyrosine-based inhibitory motif, KTIM. Consistent with PLC-?3- and SHP-1-mediated repression of Lyn activity by dephosphorylation at Tyr396, Fc?RI-mediated phenotypes were similar in Plcb3(-/-) and SHP-1 mutant mast cells. Thus, we have defined a PLC-?3- and SHP-1-mediated signaling pathway for Fc?RI-mediated cytokine production.
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Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein.
J. Biol. Chem.
PUBLISHED: 11-02-2010
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Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/?-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not ?-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.
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Pathway-selective suppression of chemokine receptor signaling in B cells by LPS through downregulation of PLC-?2.
Cell. Mol. Immunol.
PUBLISHED: 09-27-2010
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Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-?2 mRNA and protein. Data obtained using PLC-?2(-/-) mice showed that the ?2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-?2 using transfection, consistent with a functional effect of downregulating PLC-?2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-?2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.
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Important roles of PI3Kgamma in osteoclastogenesis and bone homeostasis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-02-2010
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G protein-coupled receptor-regulated PI3Kgamma is abundantly expressed in myeloid cells and has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in bone homeostasis has not been investigated, despite the fact that osteoclasts are derived from myeloid lineage. We therefore carried out thorough bone phenotypic characterization of a PI3Kgamma-deficient mouse line and found that PI3Kgamma-deficient mice had high bone mass. Our analyses further revealed that PI3Kgamma deficiency did not affect bone formation because no significant changes in osteoblast number and bone formation rate were observed. Instead, the lack of PI3Kgamma was associated with decreased bone resorption, as evidenced by decreased osteoclast number in vivo and impaired osteoclast formation in vitro. The decreased osteoclast formation was accompanied by down-regulated expression of osteoclastogenic genes, compromised chemokine receptor signaling, and an increase in apoptosis during osteoclast differentiation. Together, these data suggest that PI3Kgamma regulates bone homeostasis by modulating osteoclastogenesis. Our study also suggests that inhibition of PI3Kgamma, which is being considered as a potential therapeutic strategy for treating chronic inflammatory disorders, may result in an increase in bone mass.
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Different roles of G protein subunits beta1 and beta2 in neutrophil function revealed by gene expression silencing in primary mouse neutrophils.
J. Biol. Chem.
PUBLISHED: 06-04-2010
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Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gbeta(1) and/or Gbeta(2) in mouse neutrophils. Knockdown of Gbeta(2) appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gbeta(1) had no significant effect. However, knockdown of both Gbeta(1) and Gbeta(2) led to significant reduction in motility and responsiveness. In addition, knockdown of Gbeta(1) but not Gbeta(2) inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gbeta(1) and Gbeta(2) in the regulation of neutrophil functions.
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[Anti-tumor activity of safflower polysaccharide (SPS) and effect on cytotoxicity of CTL cells, NK cells of T739 lung cancer in mice].
Zhongguo Zhong Yao Za Zhi
PUBLISHED: 04-17-2010
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To study the anti-tumor activity of SPS in vivo and in vitro and the cytotoxicity of CTL cells, NK cells of T739 lung cancer in mice.
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Synaptotagmin-mediated vesicle fusion regulates cell migration.
Nat. Immunol.
PUBLISHED: 04-12-2010
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Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.
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Integrin-induced PIP5K1C kinase polarization regulates neutrophil polarization, directionality, and in vivo infiltration.
Immunity
PUBLISHED: 03-04-2010
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Neutrophils are important in innate immunity and acute inflammatory responses. However, the regulation of their recruitment to sites of inflammation has not been well characterized. Here, we investigated the kinase PIP5K1C and showed that PIP5K1C deficiency impaired neutrophil recruitment because of an adhesion defect. PIP5K1C regulated the adhesion through facilitating RhoA GTPase and integrin activation by chemoattractants. Integrins could induce polarization of an isoform of PIP5K1C, PIP5K1C-90, in neutrophils through intracellular vesicle transport independently of exogenous chemoattractant. PIP5K1C-90 polarization was required for polarized RhoA activation at uropods and provided an initial directional cue for neutrophil polarization on the endothelium. Importantly, the polarization was also required for circumventing the inhibition of lamellipodium formation by RhoA so that neutrophils could form leading edges required for transendothelial migration. Because integrins are not known to regulate neutrophil polarization, our study revealed a previously underappreciated role of integrin signaling in neutrophil regulation.
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Suppression of PLCbeta2 by endotoxin plays a role in the adenosine A(2A) receptor-mediated switch of macrophages from an inflammatory to an angiogenic phenotype.
Am. J. Pathol.
PUBLISHED: 10-22-2009
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Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A(2A) receptor (A(2A)R) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A(2A)Rs by TLR agonists, down-regulation of tumor necrosis factor alpha (TNFalpha) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)beta1 and beta2 expression in macrophages by de-stabilizing their mRNAs. The PLCbeta inhibitor U73122 down-regulates TNFalpha expression by macrophages, and in the presence of A(2A)R agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A(2A)R agonists. Selective down-regulation of PLCbeta2, but not PLCbeta1, using small-interfering RNA resulted in increased VEGF expression in response to A(2A)R agonists, but did not suppress TNFalpha expression. Macrophages from PLCbeta2(-/-) mice also expressed increased VEGF in response to A(2A)R agonists. LPS-mediated suppression of PLCbeta1 and beta2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 microg/mouse, i.p.) suppressed PLCbeta1 and beta2 expression in spleen, liver, and lung of wild-type but not MyD88(-/-) mice. These studies indicate that LPS suppresses PLCbeta1 and beta2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCbeta2 plays an important role in switching M1 macrophages into an M2-like state.
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Identification of triazinoindol-benzimidazolones as nanomolar inhibitors of the Mycobacterium tuberculosis enzyme TDP-6-deoxy-d-xylo-4-hexopyranosid-4-ulose 3,5-epimerase (RmlC).
Bioorg. Med. Chem.
PUBLISHED: 10-13-2009
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High-throughput screening of 201,368 compounds revealed that 1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one (SID 7975595) inhibited RmlC a TB cell wall biosynthetic enzyme. SID 7975595 acts as a competitive inhibitor of the enzymes substrate and inhibits RmlC as a fast-on rate, fully reversible inhibitor. An analog of SID 7975595 had a K(i) of 62nM. Computer modeling showed that the binding of the tethered two-ringed system into the active site occurred at the thymidine binding region for one ring system and the sugar region for the other ring system.
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GSK3: a multifaceted kinase in Wnt signaling.
Trends Biochem. Sci.
PUBLISHED: 09-10-2009
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GSK3 is one of the few signaling mediators that play central roles in a diverse range of signaling pathways, including those activated by Wnts, hedgehog, growth factors, cytokines, and G protein-coupled ligands. Although the inhibition of GSK3-mediated beta-catenin phosphorylation is known to be the key event in Wnt-beta-catenin signaling, the mechanisms that underlie this event remain incompletely understood. The recent demonstration of GSK3 involvement in Wnt receptor phosphorylation illustrates the multifaceted roles that GSK3 plays in Wnt-beta-catenin signaling. In this review, we will summarize these recent results and offer explanations, hypotheses, and models to reconcile some of these observations.
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Cell stimulation with optically manipulated microsources.
Nat. Methods
PUBLISHED: 09-02-2009
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Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 microm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.
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Regulation of immature dendritic cell migration by RhoA guanine nucleotide exchange factor Arhgef5.
J. Biol. Chem.
PUBLISHED: 08-27-2009
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There are a large number of Rho guanine nucleotide exchange factors, most of which have no known functions. Here, we carried out a short hairpin RNA-based functional screen of Rho-GEFs for their roles in leukocyte chemotaxis and identified Arhgef5 as an important factor in chemotaxis of a macrophage phage-like RAW264.7 cell line. Arhgef5 can strongly activate RhoA and RhoB and weakly RhoC and RhoG, but not Rac1, RhoQ, RhoD, or RhoV, in transfected human embryonic kidney 293 cells. In addition, Gbetagamma interacts with Arhgef5 and can stimulate Arhgef5-mediated activation of RhoA in an in vitro assay. In vivo roles of Arhgef5 were investigated using an Arhgef-5-null mouse line. Arhgef5 deficiency did not affect chemotaxis of mouse macrophages, T and B lymphocytes, and bone marrow-derived mature dendritic cells (DC), but it abrogated MIP1alpha-induced chemotaxis of immature DCs and impaired migration of DCs from the skin to lymph node. In addition, Arhgef5 deficiency attenuated allergic airway inflammation. Therefore, this study provides new insights into signaling mechanisms for DC migration regulation.
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Structure-Based Design, Synthesis, and Evaluation of 2-(2-Hydroxyethyl)-2-deoxyadenosine and the 5-Diphosphate Derivative as Ribonucleotide Reductase Inhibitors.
ChemMedChem
PUBLISHED: 08-15-2009
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Analysis of the recently solved X-ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2-hydroxy group of the nucleoside ribose. In this study 2-(2-hydroxyethyl)-2-deoxyadenosine 1 and the 5-diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2, and the co-crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized.
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Evaluation of analogs of reutericyclin as prospective candidates for treatment of staphylococcal skin infections.
Antimicrob. Agents Chemother.
PUBLISHED: 07-06-2009
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The potential for reutericyclin derivatives to be used as topical antibiotics to treat staphylococcal skin infections was investigated. All reutericyclins inhibited the growth of clinical isolates of drug-resistant Staphylococcus aureus. Unlike the standard topical agent mupirocin, most reutericyclin derivatives eradicated staphylococcal biofilms. Moreover, two compounds formulated in hydrophilic petrolatum (10%, wt/wt) were efficacious in treating S. aureus superficial skin infections in mice. These data exemplify the prospect of developing reutericyclins as new topical antibiotics.
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Regulation of phosphatidylinositol kinases and metabolism by Wnt3a and Dvl.
J. Biol. Chem.
PUBLISHED: 06-26-2009
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Wnt signaling plays important roles in various physiological and pathophysiological processes. The pathway that leads to beta-catenin stabilization is initiated by Wnt binding to its cell surface receptors, which induces the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) via activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) type I. Here, we show that Wnt also stimulated the production of phosphatidylinositol 4-phosphate (PtdIns(4)P), which depended on Frizzled (Fz), Dishevelled (Dvl), and phosphatidylinositol 4-kinase (PI4K) type II alpha in HEK293T cells. Dvl directly interacted with and activated PI4KII alpha by increasing its V(max) for ATP and PtdIns. In addition, Dvl regulated PI4KII alpha and PIP5KI via different domains. Moreover, Dvl, PI4KII alpha, and PIP5KI appeared to form a ternary complex upon Wnt3a stimulation. This complex may allow efficient production of PtdIns(4,5)P(2) from PtdIns, which is far more abundant than PtdIns(4)P in cells. Therefore, this study provides new insights into the mechanism by which Wnt3a regulates the production of PtdIns(4,5)P(2).
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Synthesis, optimization and structure-activity relationships of 3,5-disubstituted isoxazolines as new anti-tuberculosis agents.
Eur J Med Chem
PUBLISHED: 05-08-2009
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In the course of the development of a potent series of nitrofuranylamide anti-tuberculosis agents, we investigated if the exceptional activity resulted in part from the isoxazoline core and if it possessed any intrinsic anti-tuberculosis activity. This led to the discovery of an isoxazoline ester with appreciable anti-tuberculosis activity. In this study we explored the anti-tuberculosis structure-activity relationship of the isoxazoline ester compound through systematic modification of the 3,5-di-substituted isoxazoline core. Two approaches were used: (i) modification of the potentially metabolically labile ester functionality at the 3 position with acids, amines, amides, reverse amides, alcohols, hydrazides, and 1,3,4-oxadiazoles; (ii) substitution of the distal benzyl piperazine ring in the 5 position of the isoxazoline ring with piperazyl-ureas, piperazyl-carbamates, biaryl systems, piperidines and morpholine. Attempts to replace the ester group at C-3 position of isoxazoline with a variety of bioisosteric head groups led to significant loss of the tuberculosis inhibition indicating that an ester is required for anti-tuberculosis activity. Optimization of the isoxazoline C-5 position produced compounds with improved anti-tuberculosis activity, most notably the piperazyl-urea and piperazyl-carbamate analogs.
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Sites of regulated phosphorylation that control K-Cl cotransporter activity.
Cell
PUBLISHED: 05-07-2009
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Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3s intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.
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Water-soluble 3,4:9,10-perylene tetracarboxylic ammonium as a high-performance fluorochrome for living cells staining.
Luminescence
PUBLISHED: 04-16-2009
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Highly water-soluble 3,4:9,10-perylene tetracarboxylic ammonium with quantitative fluorescence quantum yield was designed. Owing to the high negative electrostatic potential of the perylene plane, the perylene dye remained stable over a broad pH range and was successfully applied as a high-performance fluorochrome for living hippocampal neurons staining.
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Tumor suppression by phospholipase C-beta3 via SHP-1-mediated dephosphorylation of Stat5.
Cancer Cell
PUBLISHED: 03-20-2009
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Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitts lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.
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Discovery, synthesis, and biological evaluation of piperidinol analogs with anti-tuberculosis activity.
Bioorg. Med. Chem.
PUBLISHED: 02-19-2009
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Direct anti-tuberculosis screening of commercially available compound libraries identified a novel piperidinol with interesting anti-tuberculosis activity and drug like characteristics. To generate a structure activity relationship about this hit a 22 member optimization library was generated using parallel synthesis. Products of this library 1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl)piperidin-4-ol and 1-((S)-3-(4-(trifluoromethyl) phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl) piperidin-4-ol demonstrated good anti-tuberculosis activity. Unfortunately, side effects were observed upon in vivo anti-tuberculosis testing of these compounds precluding their further advancement, which may be in part due to the secondary pharmacology associated with the aryl piperidinol core.
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Microwave-assisted synthesis of macrocycles via intramolecular and/or bimolecular Ullmann coupling.
Tetrahedron Lett.
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Microwave-assisted synthesis of macrocyclic diaryl ethers via intramolecular and/or bimolecular Ullmann coupling is described. Using the optimized conditions, a panel of macrocycles, with different substitution patterns, ring sizes, and linkers, has been successfully synthesized using microwave irradiation. To the best of our knowledge, this work represents the first examples of the microwave-assisted synthesis of macrocyclic diaryl ethers via intramolecular and/or bimolecular Ullmann coupling.
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Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase and NF?B inhibitors.
Org. Biomol. Chem.
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Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NF?B could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and cancer. During the course of evaluating the biological potential of a commercial compound library, 2-phenylindole (1) displayed inhibitory activity against nitrite production and NF?B with IC(50) values of 38.1 ± 1.8 and 25.4 ± 2.1 ?M, respectively. Based on this lead, synthesis and systematic optimization have been undertaken in an effort to find novel and more potent nitric oxide synthase and NF?B inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and 2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and 3-oxime derivatives 2–7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited the strongest nitrite inhibitory activities (IC(50) = 4.4 ± 0.5 and 4.8 ± 0.4 ?M, respectively); as well as NF?B inhibition (IC(50) = 6.9 ± 0.8 and 8.5 ± 2.0 ?M, respectively). In addition, the 6?-MeO-naphthalen-2?-yl indole derivative 10at displayed excellent inhibitory activity against NF?B with an IC(50) value of 0.6 ± 0.2 ?M.
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Synthesis and structure-activity relationships of lansine analogues as antileishmanial agents.
ChemMedChem
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Clear and rational thinking: A series of rationally designed, lansine-derived carbazoles was synthesized and evaluated for activity against promastigotes and amastigotes of Leishmania donovani, the causative agent of leishmaniasis. Some structural modifications gave rise to compounds with enhanced activity and selectivity over lansine, allowing structure-activity relationships to be elucidated and providing a foundation for the further development of this pharmacophore.
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A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability.
Circ. Res.
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The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity.
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Evaluation of flavonoid and resveratrol chemical libraries reveals abyssinone II as a promising antibacterial lead.
ChemMedChem
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Lead on! In the course of screening flavonoid and resveratrol libraries, abyssinone II, a naturally occurring prenylated flavonoid, was found to exhibit relatively good antitubercular and antibacterial activity. Preliminary mechanistic studies revealed that abyssinone II hyperpolarizes the bacterial membrane potential and inhibits the biosynthesis of key cellular macromolecules (DNA, RNA, and protein).
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Layered double hydroxides as flame retardant and thermal stabilizer for polymers.
Recent Pat Nanotechnol
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Layered double hydroxides (LDH) has wide applications as non-toxic and halogen-free flame retardant for various resins and highly efficient thermal stabilizer for halogen-containing polymers. This review will discuss some public patents and relevant papers on the flame retardancy and the thermal stability of LDH/polymer composites when the LDHs with different chemical compositions are used as the additive in the polymer matrix. We have summarized these related LDHs in two tables: one for flame retardant and the other for thermal stabilizer.
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Synthesis and applications of layered double hydroxides based pigments.
Recent Pat Nanotechnol
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Layered double hydroxides (LDHs) have attracted a great deal of attention owing to their structural anisotropy, anion-exchange capability and compositional flexibility and have been widely investigated as catalysts, adsorbents, anion-exchangers, polymer additives, optical materials, and so on. The intercalation of chromophores into the interlayer galleries of LDHs has drawn considerable interest since it can result in a kind of functional pigments showing different photophysical and photochemical properties from the pristine chromophores due to the host-guest and guest-guest interactions. This paper reviews recent patents progress made for the synthesis and applications of LDHs based pigments. The potentional applications and the future development are also discussed.
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Chemical and genetic evidence for the involvement of Wnt antagonist Dickkopf2 in regulation of glucose metabolism.
Proc. Natl. Acad. Sci. U.S.A.
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Mutations in Wnt receptor LRP5/6 and polymorphism in Wnt-regulated transcription factor TCF7L2 are associated with dysregulation of glucose metabolism. However, it is not clear whether Wnt antagonist Dickkopf (Dkk) has a significant role in the regulation of glucose metabolism. Here, we identified small-molecule inhibitors of Wnt antagonist Dkk through molecular modeling, computation-based virtual screens, and biological assays. One of the Dkk inhibitors reduced basal blood-glucose concentrations and improved glucose tolerance in mice. This Dkk inhibitor appeared to act through DKK2 because the inhibitor exerted no additional effects on glucose metabolism in the Dkk2(-/-) mice. Our study of Dkk2(-/-) mice showed that DKK2 deficiency was associated with increased hepatic glycogen accumulation and decreased hepatic glucose output. DKK2 deficiency did not cause in increase in insulin production but resulted in increased Wnt activity and GLP1 production in the intestines. Given that the Dkk inhibitor improved glucose tolerance in a murine model of type 2 diabetes (db/db), we suggest that DKK2 may be a potential therapeutic target for treating type 2 diabetes.
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A model of GAG/MIP-2/CXCR2 interfaces and its functional effects.
Biochemistry
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MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 Å resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.
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PRR5L degradation promotes mTORC2-mediated PKC-? phosphorylation and cell migration downstream of G?12.
Nat. Cell Biol.
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Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-? hydrophobic motif phosphorylation. The late phase is mediated by G?(12), which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-?, but not AKT, results in PKC-? hydrophobic motif phosphorylation and activation. This G?(12)-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.
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The structural basis of DKK-mediated inhibition of Wnt/LRP signaling.
Sci Signal
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Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) mediate canonical Wnt-?-catenin signaling by forming a complex with the co-receptor Frizzled, which binds to Wnt proteins. Dickkopf (DKK)-related proteins inhibit the Wnt signaling pathway by directly binding to the ectodomains of LRP5/6. However, the mechanism for DKK-mediated antagonism has not been fully understood as of yet. Crystal structures of the LRP6 ectodomain in complex with DKK1, along with mutagenesis studies, provide considerable insights into the molecular basis for DKK-mediated inhibition and Wnt signaling through LRP5/6.
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Identification Of Small Molecule TRABID Deubiquitinase Inhibitors By Computation-Based Virtual Screen.
BMC Chem Biol
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Wnt/?-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where ?-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/?-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.