Shape-Complementary Multicomponent Assembly of Low-Symmetry Co(III)Salphen-Based Coordination Cages.

While metal-mediated self-assembly is a popular technique to construct discrete nanosized objects, highly symmetric structures, built from one type of ligand at a time, are dominating reported systems. The tailored integration of a set of different ligands requires sophisticated approaches to avoid narcissistic separation or formation of statistical mixtures. Here, we demonstrate how the combination of three structure-guiding effects (metal-templated macrocyclization, additional bridging ligands and shape-complementarity) based on Co(III)salphen metal nodes allows for a rational and high-yielding synthesis of structurally complex, lantern-shaped cages with up to four differentiable bridges. Three new heteroleptic coordination cages based on dinuclear Co(III)salphen macrocycles were synthesized in a one-pot reaction approach and fully characterized, including single crystal X-ray analyses. One cage groups two of the same ligands, another two different ligands around a symmetric Co2-bis-salphen ring. In the most complex structure, this ring is unsymmetric, rendering all four connections between the two metal centers distinguishable. While heteroleptic assembly around Pd(II) nodes has been shown to be dynamic, beneficial for cage-to-cage transformations, assembly cascades and adaptive systems, the herein introduced cages based on kinetically more inert Co(III)salphen will be advantageous for applications in enzyme-like catalysis and molecular machinery that require enhanced structural and chemical stability.

Crystals of Ni6L12 Ellipsoidal Tubes as Single-Crystal-to-Single-Crystal Adsorption Matrix: Penetrative Study of Self-Assembled Crystals vs Guest-Exchanged Crystals.

Informative similarities/differences between self-assembled and single-crystal-to-single-crystal (SCSC) guest-exchanged crystals based on both the molecular structure and adsorption nature are observed. The self-assembly of Ni(ClO4)2 with a dicyclopentyldi(pyridine-3-yl)silane bidentate ligand (L) in a mixture of toluene and acetonitrile gives rise to purple crystals consisting of double-stranded ellipsoidal tubes, [Ni6(ClO4)4(CH3CN)8L12]·8ClO4·4CH3CN·5C7H8. The coordinated acetonitriles as well as the solvates are removed at 170 °C to transform the purple crystals into blue crystals of [Ni(ClO4)2L2]n that return to the original crystals in the mixture of toluene and acetonitrile. Further, the toluene and acetonitrile solvates of the original crystals are replaced by o-, m-, and p-xylene isomers within 5 min in a SCSC manner. In the present study, SCSC xylene-exchanged crystals were compared with crystals obtained from direct self-assembly in a mixture of each xylene isomer and acetonitrile.

iPSC-Derived MSCs Are a Distinct Entity of MSCs with Higher Therapeutic Potential than Their Donor-Matched Parental MSCs.

Mesenchymal stromal cells derived from induced pluripotent stem cells (iMSCs) have been proposed as alternative sources of primary MSCs with various advantages for cell therapeutic trials. However, precise evaluation of the differences between iMSCs and primary MSCs is lacking due to individual variations in the donor cells, which obscure direct comparisons between the two. In this study, we generated donor-matched iMSCs from individual bone marrow-derived MSCs and directly compared their cell-autonomous and paracrine therapeutic effects. We found that the transition from primary MSCs to iMSCs is accompanied by a functional shift towards higher proliferative activity, with variations in differentiation potential in a donor cell-dependent manner. The transition from MSCs to iMSCs was associated with common changes in transcriptomic and proteomic profiles beyond the variations of their individual donors, revealing expression patterns unique for the iMSCs. These iMSC-specific patterns were characterized by a shift in cell fate towards a pericyte-like state and enhanced secretion of paracrine cytokine/growth factors. Accordingly, iMSCs exhibited higher support for the self-renewing expansion of primitive hematopoietic progenitors and more potent immune suppression of allogenic immune responses than MSCs. Our study suggests that iMSCs represent a separate entity of MSCs with unique therapeutic potential distinct from their parental MSCs, but points to the need for iMSC characterization in the individual basis.

Light-Powered Dissipative Assembly of Diazocine Coordination Cages.

Stimuli-responsive coordination cages allow reversible control over guest binding and release, relevant for adaptive receptors, carriers, catalysts, and complex systems. Light serves as an advantageous stimulus, as it can be applied with precise spatial and temporal resolution without producing chemical waste products. We report the first Pd-mediated coordination cage based on ligands embedding a diazocine photoswitch. While the thermodynamically more stable cis-photoisomer sloppily assembles to a mixture of species with general formula [Pdncis-L2n], the less stable trans-isomer yields a defined [Pd2trans-L4] cage that reversibly converts back to the cis-system by irradiation at 530 nm or thermal relaxation. The [Pdncis-L2n] species do not bind a given guest; however, [Pd2trans-L4] is able to encapsulate a bis-sulfonate as long as it is kept assembled, requiring continuous irradiation at 385 nm. In the absence of UV light, thermal relaxation results in back-switching and guest release. Assembly and properties of the system were characterized by a combination of NMR, ion mobility ESI-MS, single-crystal X-ray diffraction, and UV-vis absorption studies.

Reversible switching of leukemic cells to a drug-resistant, stem-like subset via IL-4-mediated cross-talk with mesenchymal stroma.

Chemoresistance of leukemic cells has largely been attributed to clonal evolution secondary to accumulating mutations. Here, we show that a subset of leukemic blasts in contact with the mesenchymal stroma undergo cellular conversion into a distinct cell type that exhibits a stem cell-like phenotype and chemoresistance. These stroma-induced changes occur in a reversible and stochastic manner driven by cross-talk, whereby stromal contact induces interleukin-4 in leukemic cells that in turn targets the mesenchymal stroma to facilitate the development of new subset. This mechanism was dependent on interleukin-4-mediated upregulation of vascular cell adhesion molecule- 1 in mesenchymal stroma, causing tight adherence of leukemic cells to mesenchymal progenitors for generation of new subsets. Together, our study reveals another class of chemoresistance in leukemic blasts via functional evolution through stromal cross-talk, and demonstrates dynamic switching of leukemic cell fates that could cause a non-homologous response to chemotherapy in concert with the patient-specific microenvironment.

Integrative Assembly of Heteroleptic Tetrahedra Controlled by Backbone Steric Bulk.

A bent fluorenone-based dipyridyl ligand LA reacts with PdII cations to a solvent-dependent dynamic library of [PdnL2n] assemblies, constituted by a [Pd3LA6] ring and a [Pd4LA8] tetrahedron as major components, and a [Pd6LA12] octahedron as minor component. Introduction of backbone steric hindrance in ligand LB allows exclusive formation of the [Pd6LB12] octahedron. Combining equimolar amounts of both ligands results in integrative self-sorting to give an unprecedented [Pd4LA4LB4] heteroleptic tetrahedron. Key to the non-statistical assembly outcome is exploiting the structural peculiarity of the [Pd4L8] tetrahedral topology, where the four lean ligands occupy two doubly bridged edges and the bulky ligands span the four remaining, singly bridged edges. Hence, the system finds a compromise between the entropic drive to form an assembly smaller than the octahedron and the enthalpic prohibition of pairing two bulky ligands on the same edge of the triangular ring. The emission of luminescent LA is maintained in both homoleptic [Pd3LA6] and heteroleptic [Pd4LA4LB4].

Multi-stimuli Control over Assembly and Guest Binding in Metallo-supramolecular Hosts Based on Dithienylethene Photoswitches.

It is difficult to assemble multi-component metallo-supramolecular architectures in a non-statistical fashion, which limits their development toward functional materials. Herein, we report a system of interconverting bowls and cages that are able to respond to various selective stimuli (light, ligands, anions), based on the self-assembly of a photochromic dithienylethene (DTE) ligand, La, with PdII cations. By combining the concept of "coordination sphere engineering", relying on bulky quinoline donors, with reversible photoswitching between the ligand's open (o-La) and closed (c-La) forms, a [Pd2(o-La)4] cage (o-C) and a [Pd2(c-La)3] bowl (c-B) were obtained, respectively. This structural rearrangement modulates the system's guest uptake capabilities. Among three bis-sulfonate guests (G1, G2, and G3), the cage can encapsulate only the smallest (G1), while the bowl binds all of them. Bowl c-B was further used to synthesize a series of heteroleptic cages, [Pd2LA3LB], representing a motif never reported before. Additional ligands (Lc-f), with short or long arms, tune the cavity size, thus enabling or preventing guest uptake. Addition of Br-/Ag+ makes it possible to change the overall charge, again triggering guest uptake and release, as well as fourth ligand de-/recomplexation. In combination, site-selective introduction of functionality and application of external stimuli lead to an intricate system of hosts with different guest preferences. A high degree of complexity is achieved through cooperativity between only a few components.

Integrin α5ß1 activates the NLRP3 inflammasome by direct interaction with a bacterial surface protein.

Integrins are cell-surface heterodimeric glycoproteins composed of alpha and beta subunits that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. In this study, we report a specific role of integrin α5ß1 in NLRP3 inflammasome activation in macrophages stimulated by Td92, a surface protein of the periodontopathogen, Treponema denticola. The direct interaction of Td92 with the cell membrane integrin α5ß1 resulted in ATP release and K(+) efflux, which are the main events in NLRP3 activation. This interaction was arginine-glycine-aspartate (RGD)-independent, and Td92 internalization was not required for the activity. An integrin α5ß1 antibody and oxATP, an ATP receptor antagonist, inhibited NLRP3 expression, caspase-1 activation, interleukin-1ß (IL-1ß) secretion, and proIL-1ß synthesis, all of which were regulated by NF-κB activation. Therefore, our data has identified the integrin α5ß1 as a principal cell membrane receptor for both NLRP3 inflammasome activation and IL-1ß transcription by a bacterial protein, which could exaggerate inflammation, a characteristic of periodontitis.

Combination of LMT-28 and Metformin Improves Beneficial Anti-Inflammatory Effect in Collagen-Induced Arthritis.

OBJECTIVES: The interleukin-6 (IL-6)-mediated signaling pathway plays an essential role in the development of rheumatoid arthritis. LMT-28 suppresses the activation of the IL-6-mediated signaling by direct targeting of gp130. Although LMT-28 and metformin both possess anti-inflammatory activity, the beneficial effect of LMT-28 and metformin combination on a collagen-induced arthritis (CIA) model has not yet been investigated. This study aimed to investigate the anti-inflammatory effect and mechanism of a combination of LMT-28 and metformin in a CIA model. METHODS: In MH7A cells, cell proliferation and the IL-6-mediated signaling pathway following administration of LMT-28 and metformin combination was analyzed through MTT assay and Western blotting. The level of T helper 17 (Th17) cell differentiation from CD4+ T cells was analyzed in mouse splenocytes and human peripheral blood mononuclear cells. Arthritis score, incidence rate, inflammatory cytokine, and T-cell subsets were measured in CIA mice following administration of LMT-28 and metformin combination. RESULTS: Combination treatment with LMT-28 and metformin diminished proliferation of MH7A cells and IL-6-mediated gp130, STAT3, and ERK signaling more than in individual treatments. Furthermore, the differentiation of CD4+ T cells into Th17 cells was attenuated more by combination treatment with LMT-28 and metformin than individual treatments. The combination of LMT-28 and metformin ameliorated the arthritic score better than individual treatments. The combination significantly reduced tumor necrosis factor and IL-6 levels in the sera and had an anti-inflammatory effect on the distribution of Treg/Th17 cells in the lymph nodes. CONCLUSION: Combination treatment with LMT-28 and metformin significantly ameliorates arthritic symptoms in CIA by suppressing Th17 differentiation and IL-6 signaling.

Comparative effectiveness of different antiplatelet agents at reducing TNF-driven inflammatory responses in a mouse model.

Antiplatelet drugs are conventionally used as treatments because of their anti-coagulation functions. However, their pleiotropic effects are of great significance to the treatment of ischaemic cardiovascular diseases. Many studies have reported that an excessive amount of inflammation driven by tumour necrosis factor (TNF) is closely related to the prevalence of atherosclerosis. As the drug selection criteria and evaluation methods related to the anti-TNF activity of antiplatelet drugs remain limited, our investigation of these drugs should prove beneficial. In this study, we compared the anti-TNF activity of three antiplatelet agents, namely clopidogrel, sarpogrelate, and cilostazol, using the TNF-induced inflammatory mouse model. After the oral administration of these drugs, acute inflammation was induced via injection of lipopolysaccharide (LPS) or D-galactosamine (D-gal) and TNF. Serum TNF levels, and the mRNA and protein expression levels of TNF in mouse heart tissue, macrophage accumulation in aortic lesions, and mouse survival were analysed to compare the anti-TNF effects of the three antiplatelet agents. Of the three antiplatelet agents, cilostazol significantly reduced the different levels under the most effective observation. In addition, cilostazol was found to attenuate the TNF-stimulated phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) p65 in the aortic vascular smooth muscle cell line, MOVAS-1 and the D-gal plus TNF-challenged heart tissue of mouse. Therefore, cilostazol is the most ideal of the three antiplatelet drugs for the treatment of TNF-mediated inflammatory disorders.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Anti-TNF effect of combined pravastatin and cilostazol treatment in an in vivo mouse model.

Objectives: Pravastatin and cilostazol are used as lipid-lowering and antiplatelet agents, respectively. Regarding their well-known anti-inflammatory effects, the additive effect of the two drugs on anti-TNF functions has not yet been investigated. In the present investigation, the beneficial effect of combined pravastatin and cilostazol on their anti-TNF activities was assessed using an in vivo mouse model. Methods: Mice were pretreated with pravastatin and/or cilostazol (40 mg/kg of each), orally once two hour prior to an LPS (5 mg/kg, i.p.) challenge. One hour post challenge, blood and descending aorta were collected for serum TNF levels and immune cell infiltration analyses. For survival analysis, pravastatin and/or cilostazol (40 mg/kg of each) were administered 30 minutes prior to d-galactosamine administration (700 mg/kg, i.p.) and TNF (10 µg/kg, i.p.) challenge and mice survival was monitored. We also examined the effect of either drug or the combination of drugs on TNF-mediated MAPK and NF-κB signaling, using Western blot analysis. Results: Combined treatment of pravastatin and cilostazol significantly decreased serum TNF release and immune cell infiltration in the descending aorta following LPS administration, compared to each single treatment. Additionally, the combined drugs significantly decreased TNF-mediated mouse mortality and downregulated TNF-induced MAPK and NF-κB activation. Conclusions: These findings suggest that combined pravastatin and cilostazol is more effective for reducing TNF-driven inflammation through their anti-TNF activity than monotherapy.

Muramyl dipeptide potentiates a Bacillus anthracis poly-γ-d-glutamic acid capsule surrogate that induces maturation and activation of mouse dendritic cells.

Poly-γ-d-glutamic acid (PGA) of anthrax is an important pathogenic factor due to its anti-phagocytic activity. Additionally, PGA has the ability to activate mouse macrophages for the secretion of cytokines through Toll-like receptor (TLR) 2. Peptidoglycan (PGN), a major bacterial cell-wall component, induces inflammatory responses in the host. We assessed whether PGA can induce maturation and cytokine expression in immature mouse dendritic cells (DCs) in the existence of muramyl dipeptide (MDP), the minimum motif of PGN with immunostimulatory activity. Stimulation of immature DCs with PGA or MDP alone augmented expression of costimulatory molecules and MHC class II proteins, which are all cell surface markers indicative of maturation. The observed effects were further enhanced by costimulation of PGA and MDP. PGA alone was sufficient to induce expression of TNF-α, IL-6, MCP-1, and MIP1-α, whereas MDP alone did not under the same conditions. Treatment with MDP enhanced PGA-induced expression of the tested inflammatory mediators; however, the synergistic effect found for PGA and MDP was not observed in TLR2- or nucleotide-binding oligomerization domain (NOD) 2-knockout DCs. Additionally, MDP augmented PGA-induced MAP kinases and NF-κB activation, which is crucial for expression of cytokines. Furthermore, MAP kinase and NF-κB inhibitors attenuated MDP enhancement of PGA-induced cytokine production. In addition, co-culture of splenocytes and PGA/MDP-matured DCs induced higher expression of IL-2 and IFN-γ compared to that of splenocytes and PGA-matured DCs. Collectively, our results suggest that PGA and MDP cooperatively induce inflammatory responses in mouse DCs through TLR2 and NOD2 via MAP kinase and NF-κB pathways, subsequently leading to lymphocyte activation.

Construction of Hetero-Four-Layered Tripalladium(II) Cyclophanes by Transannular π⋠⋠⋠π Interactions.

A synthetic strategy for the generation of new molecular species utilizing a provision of nature is presented. Nano-dimensional (23(2)×21(1)×16(1) ų) hetero-four-layered trimetallacyclophanes were constructed by proof-of-concept experiments that utilize a suitable combination of π⋅⋅⋅π interactions between the central aromatic rings, tailor-made short/long spacer tridentate donors, and the combined helicity. The behavior of the unprecedented four-layered metallacyclophane system offers a landmark in the development of new molecular systems.

The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist.

Bacillus anthracis is a pathogenic Gram-positive bacterium that causes a highly lethal infectious disease, anthrax. The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of B. anthracis, along with exotoxins. PGA enables B. anthracis to escape phagocytosis and immune surveillance. Our previous study showed that PGA activates the human macrophage cell line THP-1 and human dendritic cells, resulting in the production of the proinflammatory cytokine interleukin-1ß (IL-1ß) (M. H. Cho et al., Infect Immun 78:387-392, 2010, http://dx.doi.org/10.1128/IAI.00956-09). Here, we investigated PGA-induced cytokine responses and related signaling pathways in mouse bone marrow-derived macrophages (BMDMs) using Bacillus licheniformis PGA as a surrogate for B. anthracis PGA. Upon exposure to PGA, BMDMs produced proinflammatory mediators, including tumor necrosis factor alpha (TNF-α), IL-6, IL-12p40, and monocyte chemoattractant protein 1 (MCP-1), in a concentration-dependent manner. PGA stimulated Toll-like receptor 2 (TLR2) but not TLR4 in Chinese hamster ovary cells expressing either TLR2 or TLR4. The ability of PGA to induce TNF-α and IL-6 was retained in TLR4(-/-) but not TLR2(-/-) BMDMs. Blocking experiments with specific neutralizing antibodies for TLR1, TLR6, and CD14 showed that TLR6 and CD14 also were necessary for PGA-induced inflammatory responses. Furthermore, PGA enhanced activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB), which are responsible for expression of proinflammatory cytokines. Additionally, PGA-induced TNF-α production was abrogated not only in MyD88(-/-) BMDMs but also in BMDMs pretreated with inhibitors of MAP kinases and NF-κB. These results suggest that immune responses induced by PGA occur via TLR2, TLR6, CD14, and MyD88 through activation of MAP kinase and NF-κB pathways.

Metformin enhances the anti-adipogenic effects of atorvastatin via modulation of STAT3 and TGF-ß/Smad3 signaling.

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases. Interactions of master transcription factors and signaling cascades are required for adipogenesis. Regulation of excessive adipogenic processes may be an attractive therapeutic for treatment of obesity and obesity-related diseases. In this study, we found that atorvastatin exerts an anti-adipogenic activity in 3T3-L1 pre-adipocytes, and that this activity is elevated in combination with metformin. Expression of the adipogenic master regulators PPARγ and C/EBPα, and their target gene aP2, was suppressed by atorvastatin. Furthermore, atorvastatin treatment resulted in increased activation of the key master regulator of cellular energy homeostasis, AMPK. These biological activities of atorvastatin were elevated in combination with metformin. These anti-adipogenic activities were associated with regulation of the STAT3 and TGF-ß signaling cascades, resulting in the regulation of the expression of STAT3 target genes, such as KLF5, p53, and cyclin D1, and TGF-ß signaling inhibitory genes, such as SMAD7. Our results suggest that combination therapy with atorvastatin and metformin may have therapeutic potential for the treatment of obesity and obesity-related diseases caused by excessive adipogenesis.

Organization and Energy Transfer of Fused Aromatic Hydrocarbon Guests within Anion-Confining Nanochannel MOFs.

The self-assembly of Zn(II) ions with 1,3,5-tris(isonicotinoyloxyethyl)cyanurate produces new topological (4(2)⋅12(4))3(4(3))4 2D metal-organic frameworks (MOFs) with anion-confining cages. The eclipsed assembly of each 2D MOF by π-π stacking of cyanurate moieties (3.352(5) Å) forms 3D MOFs consisting of nanochannels (10.5 Å). Two of the three anions are confined in each peanut-type cage, resulting in hydrophobicity of the nanochannels. The hydrophobic nanochannel effectively adsorbs a wide range of fused aromatic hydrocarbons (FAHs) as monomers or dimers, rendering it potentially highly useful as an energy-transfer material.

Monoclonal antibody against the poly-gamma-D-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge.

BACKGROUND: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. METHODS: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. RESULTS: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. GENERAL SIGNIFICANCE: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.

BACKGROUND: Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis. RESULTS: We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody. CONCLUSIONS: Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

Coordinating nature of M6L12 double-stranded macrocycles: co-ligand competition of perchlorate, water, and acetonitrile depending on metal(II) ions.

Self-assembly of M(ClO4)2 (M(II) = Mn(II), Co(II), Ni(II), Cu(II), and Zn(II)) with dicyclopentyldi(pyridine-3-yl)silane (L) as a donor in a mixture of acetonitrile and toluene produces crystals consisting of M6L12 double-stranded macrocycles. The geometry around the M(II) cations is a typical octahedral arrangement, but the metallamacrocycles' outer axial coordination environment is sensitive to the M(II) cations. The conformation of the unique metallamacrocycles is informatively dependent on the nature of the coordination around the M(II) cations via subtle co-ligand competition among perchlorate anions, water, and acetonitrile. Both the coordinated acetonitriles and the solvate molecules of the crystals are removed at 170 °C, thereby transforming the crystals into new crystals that return to their original form in the mixture of toluene and acetonitrile. Catalytic oxidation of 3,5-di-tert-butylcatechol using [Cu6(ClO4)8(CH3CN)4L12]4ClO4·5C7H8 is much faster than those using the transformed product, [Cu(ClO4)2L2], and a simple mixture of Cu(ClO4)2 + L.