The dual role of DksA protein in the regulation of Escherichia coli pArgX promoter.

Gene expression regulation by the stringent response effector, ppGpp, is facilitated by DksA protein; however DksA and ppGpp can play independent roles in transcription. In Escherichia coli, the pArgX promoter which initiates the transcription of four tRNA genes was shown to be inhibited by ppGpp. Our studies on the role of DksA in pArgX regulation revealed that it can stimulate transcription by increasing the binding of RNA polymerase to the promoter and the productive transcription complex formation. However, when DksA is present together with ppGpp a severe down-regulation of promoter activity is observed. Our results indicate that DksA facilitates the effects of ppGpp to drive formation of inactive dead-end complexes formed by RNA polymerase at the ArgX promoter. In vivo, ppGpp-mediated regulation of pArgX transcription is dependent on DksA activity. The potential mechanisms of opposing pArgX regulation by ppGpp and DksA are discussed. pArgX is the first reported example of the promoter stimulated by DksA and inhibited by ppGpp in vitro when an overall inhibition occurs in the presence of both regulators. A dual role is thus proposed for DksA in the regulation of the pArgX promoter activity.

Magnolia officinalis L. Fortified Gum Improves Resistance of Oral Epithelial Cells Against Inflammation.

Inflammatory diseases of the periodontal tissues are known health problems worldwide. Therefore, anti-inflammatory active compounds are used in oral care products to reduce long-term inflammation. In addition to inducing inflammation, pathogen attack leads to an increased production of reactive oxygen species (ROS), which may lead to oxidative damage of macromolecules. Magnolia officinalis L. bark extract (MBE) has been shown to possess antioxidant and anti-inflammatory potential in vitro. In the present study, the influence of MBE-fortified chewing gum on the resistance against lipopolysaccharide (LPS)-induced inflammation and oxidative stress of oral epithelial cells was investigated in a four-armed parallel designed human intervention trial with 40 healthy volunteers. Ex vivo stimulation of oral epithelial cells with LPS from Porphyromonas gingivalis for 6[Formula: see text]h increased the mRNA expression and release of the pro-inflammatory cytokines IL-1[Formula: see text], IL-[Formula: see text], IL-8, MIP-1[Formula: see text], and TNF[Formula: see text]. Chewing MBE-fortified gum for 10[Formula: see text]min reduced the ex vivo LPS-induced increase of IL-8 release by 43.8 [Formula: see text] 17.1% at the beginning of the intervention. In addition, after the two-week intervention with MBE-fortified chewing gum, LPS-stimulated TNF[Formula: see text] release was attenuated by 73.4 [Formula: see text] 12.0% compared to chewing regular control gum. This increased resistance against LPS-induced inflammation suggests that MBE possesses anti-inflammatory activity in vivo when added to chewing gum. In contrast, the conditions used to stimulate an immune response of oral epithelial cells failed to induce oxidative stress, measured by catalase activity, or oxidative DNA damage.

Potential benefits of chewing gum for the delivery of oral therapeutics and its possible role in oral healthcare.

INTRODUCTION: Over the years, chewing gum has developed from a candy towards an oral health-promoting nutraceutical. This review summarizes evidence for the oral health benefits of chewing gum, emphasizing identification of active ingredients in gum that facilitate prevention and removal of oral biofilm. AREAS COVERED: Chewing of sugar-free gum yields oral health benefits that include clearance of food debris, reduction in oral dryness, increase of biofilm pH and remineralization of enamel. These basic effects of chewing gum are attributed to increased mastication and salivation. Active ingredients incorporated in chewing gums aim to expand these effects to inhibition of extrinsic tooth stain and calculus formation, enhanced enamel remineralization, reduction of the numbers of bacteria in saliva and amount of oral biofilm, neutralization of biofilm pH, and reduction of volatile sulfur compounds. EXPERT OPINION: Evidence for oral-health benefits of chewing gum additives is hard to obtain due to their relatively low concentrations and rapid wash-out. Clinical effects of gum additives are overshadowed by effects of increased mastication and salivation due to the chewing of gum and require daily chewing of gum for prolonged periods of time. Future studies on active ingredients should focus on specifically targeting pathogenic bacteria, whilst leaving the healthy microbiome unaffected.

Molecular basis of Streptococcus mutans sortase A inhibition by the flavonoid natural product trans-chalcone.

Sortase A (SrtA) from Gram positive pathogens is an attractive target for inhibitors due to its role in the attachment of surface proteins to the cell wall. We found that the plant natural product trans-chalcone inhibits Streptococcus mutans SrtA in vitro and also inhibited S. mutans biofilm formation. Mass spectrometry revealed that the trans-chalcone forms a Michael addition adduct with the active site cysteine. The X-ray crystal structure of the SrtA H139A mutant provided new insights into substrate recognition by the sortase family. Our study suggests that chalcone flavonoids have potential as sortase-specific oral biofilm inhibitors.

Quantification and qualification of bacteria trapped in chewed gum.

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 10(8) bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.

DNA-encapsulated magnesium phosphate nanoparticles elicit both humoral and cellular immune responses in mice.

The efficacy of pEGFP (plasmid expressing enhanced green fluorescent protein)-encapsulated PEGylated (meaning polyethylene glycol coated) magnesium phosphate nanoparticles (referred to as MgPi-pEGFP nanoparticles) for the induction of immune responses was investigated in a mouse model. MgPi-pEGFP nanoparticles induced enhanced serum antibody and antigen-specific T-lymphocyte responses, as well as increased IFN-? and IL-12 levels compared to naked pEGFP when administered via intravenous, intraperitoneal or intramuscular routes. A significant macrophage response, both in size and activity, was also observed when mice were immunized with the nanoparticle formulation. The response was highly specific for the antigen, as the increase in interaction between macrophages and lymphocytes as well as lymphocyte proliferation took place only when they were re-stimulated with recombinant green fluorescence protein (rGFP). Thus the nanoparticle formulation elicited both humoral as well as cellular responses. Cytokine profiling revealed the induction of Th-1 type responses. The results suggest DNA-encapsulated magnesium phosphate (MgPi) nanoparticles may constitute a safer, more stable and cost-efficient DNA vaccine formulation.

Identification of Magnolia officinalis L. bark extract as the most potent anti-inflammatory of four plant extracts.

This study was designed to compare the anti-inflammatory potential of a Magnolia officinalis L. bark extract solely or in combination with extracts prepared from either Polygonum aviculare L., Sambucus nigra L., or Isodon japonicus L. in bacterial lipopolysaccharide (LPS) stimulated human gingival fibroblasts (HGF-1) and human U-937 monocytes, as cell models of periodontal disease. HGF-1 and U-937 cells were incubated with LPS from either Porphyromonas gingivalis or Escherichia coli together with the four plant extracts alone or in combination. Secretion of anti-inflammatory cytokines from HGF-1 and U-937 cells was measured by means of a multiplexed bead assay system. Magnolia officinalis L. bark extract, at concentrations of 1 µg/mL and 10 µg/mL, reduced interleukin 6 (IL-6) and interleukin-8 (IL-8) secretion from HGF-1 cells to 72.5 ± 28.6% and reduced matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) secretion from U-937 cells to 8.87 ± 7.97% compared to LPS-treated cells (100%). The other three extracts also reduced secretion of these inflammatory markers but were not as effective. Combination of 9 µg/mL Magnolia officinalis L. extract with 1 µg/mL of each of the other extracts maintained the anti-inflammatory effect of Magnolia officinalis L. extract. Combination of 5 µg/mL Magnolia officinalis L. extract with 5 µg/mL Isodon japonicus L. extract also maintained the anti-inflammatory potential of the Magnolia officinalis L. extract, whereas increasing concentrations of any of the other plant extracts in the combination experiments reduced the Magnolia officinalis L. extract efficacy in U-937 cells.

A composite polymer nanoparticle overcomes multidrug resistance and ameliorates doxorubicin-associated cardiomyopathy.

Acquired chemotherapy resistance is a major contributor to treatment failure in oncology. For example, the efficacy of the common anticancer agent doxorubicin (DOX) is limited by the emergence of multidrug resistance (MDR) phenotype in cancer cells. While dose escalation of DOX can circumvent such resistance to a degree, this is precluded by the appearance of cardiotoxicity, a particularly debilitating condition in children. In vitro studies have established the ability of the natural phytochemical curcumin to overcome MDR; however, its widespread clinical application is restricted by poor solubility and low bioavailability. Building upon our recently developed polymer nanoparticle of curcumin (NanoCurc or NC) that significantly enhances the systemic bioavailability of curcumin, we synthesized a doxorubicin-curcumin composite nanoparticle formulation called NanoDoxCurc (NDC) for overcoming DOX resistance. Compared to DOX alone, NDC inhibited the MDR phenotype and caused striking growth inhibition both in vitro and in vivo in several models of DOX-resistant cancers (multiple myeloma, acute leukemia, prostate and ovarian cancers, respectively). Notably, NDC-treated mice also demonstrated complete absence of cardiac toxicity, as assessed by echocardiography, or any bone marrow suppression, even at cumulative dosages where free DOX and pegylated liposomal DOX (Doxil®) resulted in demonstrable attenuation of cardiac function and hematological toxicities. This improvement in safety profile was achieved through a reduction of DOX-induced intracellular oxidative stress, as indicated by total glutathione levels and glutathione peroxidase activity in cardiac tissue. A composite DOX-curcumin nanoparticle that overcomes both MDR-based DOX chemoresistance and DOX-induced cardiotoxicity holds promise for providing lasting and safe anticancer therapy.

A polymeric nanoparticle formulation of curcumin (NanoCurc™) ameliorates CCl4-induced hepatic injury and fibrosis through reduction of pro-inflammatory cytokines and stellate cell activation.

Plant-derived polyphenols such as curcumin hold promise as a therapeutic agent in the treatment of chronic liver diseases. However, its development is plagued by poor aqueous solubility resulting in poor bioavailability. To circumvent the suboptimal bioavailability of free curcumin, we have developed a polymeric nanoparticle formulation of curcumin (NanoCurc™) that overcomes this major pitfall of the free compound. In this study, we show that NanoCurc™ results in sustained intrahepatic curcumin levels that can be found in both hepatocytes and non-parenchymal cells. NanoCurc™ markedly inhibits carbon tetrachloride-induced liver injury, production of pro-inflammatory cytokines and fibrosis. It also enhances antioxidant levels in the liver and inhibits pro-fibrogenic transcripts associated with activated myofibroblasts. Finally, we show that NanoCurc™ directly induces stellate cell apoptosis in vitro. Our results suggest that NanoCurc™ might be an effective therapy for patients with chronic liver disease.

Multifunctional silica nanoparticles with potentials of imaging and gene delivery.

This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA onto it. The silica NP's surface was modified by 3-aminopropyltrimethoxysilane for DNA to bind electrostatically. Silica NPs with low polydispersity and encapsulating gadolinium oxide were prepared in the aqueous core of the reverse micelles. The average size of these spherical silica NPs doped with gadolinium oxide and dispersed in water is ∼ 50 nm as measured by dynamic light scattering and transmission electron microscopy. The plasmid DNA electrostatically held over NP's surface was firmly immobilized and protected from DNase attack. The gadolinium oxide-doped silica NPs are paramagnetic as observed from the nuclear magnetic resonance (NMR) line-broadening effect on proton spectrum of the surrounding water. In vitro transfection efficiencies of these gadolinium oxide-doped and DNA-conjugated silica NPs in COS-7 and 293T cells were found to be about 75% and 77% respectively of that of 'Polyfect®' as positive control. FROM THE CLINICAL EDITOR: This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA. These NPs are paramagnetic with in vitro transfection efficiencies in COS-7 and 293T cells of about 75% and 77% compared to 'Polyfect®' as positive control.

Neuroprotective and neurorescue effects of a novel polymeric nanoparticle formulation of curcumin (NanoCurc™) in the neuronal cell culture and animal model: implications for Alzheimer's disease.

Alzheimer's disease (AD) is characterized by deposition of amyloid-ß (Aß) plaques within the brain parenchyma followed by synaptic loss and neuronal death. Deposited Aß reacts with activated microglia to produce reactive oxygen species (ROS) and cytochemokines, which lead to severe neuroinflammation. Curcumin is a yellow polyphenol compound found in turmeric, a widely used culinary ingredient that possesses anti-inflammatory and anti-cancer properties and may show efficacy as a potential therapeutic agent in several neuro-inflammatory diseases including AD. However, poor aqueous solubility and sub-optimal systemic absorption from the gastrointestinal tract may represent factors contributing to its failure in clinical trials. To increase curcumin's bioavailability, a polymeric nanoparticle encapsulated curcumin (NanoCurc™) was formulated which is completely water soluble. NanoCurc™ treatment protects neuronally differentiated human SK-N-SH cells from ROS (H2O2) mediated insults. NanoCurc™ also rescues differentiated human SK-N-SH cells, which were previously insulted with H2O2. In vivo, intraperitoneal (IP) NanoCurc™ injection at a dose of 25mg/kg twice daily in athymic mice resulted in significant curcumin levels in the brain (0.32 µg/g). Biochemical study of NanoCurc™-treated athymic mice revealed decreased levels of H2O2 as well as caspase 3 and caspase 7 activities in the brain, accompanied by increased glutathione (GSH) concentrations. Increased free to oxidized glutathione (GSH:GSSH) ratio in athymic mice brain versus controls also indicated a favorable redox intracellular environment. Taken together, these results suggest that NanoCurc™ represents an optimized formulation worthy of assessing the therapeutic value of curcumin in AD.

Antileishmanial activity of nanocurcumin.

BACKGROUND: Leishmaniasis is an endemic disease having a wide spectrum ranging from visceral, cutaneous and mucocutaneous forms caused by unicellular, obligate intracellular parasites of the monocyte-macrophage system. The aim of the present study was to develop an effective, nontoxic and biodegradable polymeric drug-delivery system encapsulating curcumin in its hydrophobic core for the treatment of visceral leishmaniasis. RESULTS: We have reported a co-polymeric micelle of N-isopropyl acrylamide, vinyl pyrrolidone and acrylic acid in 85:10:5 M ratios through free radical polymerization. The characterization of curcumin-loaded nanoparticles (40-50 nm) was done by transmission electron microscopy, dynamic light scattering and spectroscopic methods such as NMR which ensures polymerization and formation of nanoparticles has been achieved. Nanocurcumin was evaluated as an antileishmanial agent through spleenomegaly and delayed-type hypersensitivity experiments. CONCLUSION: Nanocurcumin has shown significantly greater in vivo therapeutic efficacy than pentamidine and free curcumin in an animal model of visceral leishmaniasis. The use of nanocurcumin compared with conventional drugs and free curcumin may prove more feasible and provide a better approach towards treatment of leishmaniasis.

SEF/IL-17R (SEFIR) is not enough: an extended SEFIR domain is required for il-17RA-mediated signal transduction.

IL-17, the hallmark cytokine of the Th17 population, mediates immunity to extracellular pathogens and promotes autoimmune immunopathology. The signaling mechanisms triggered by the IL-17 receptor (IL-17RA) and related receptors are strikingly different from other cytokine subclasses. Namely, IL-17Rs contain a conserved SEF/IL-17R (SEFIR) subdomain that engages Act1, leading to activation of TRAF6, NF-κB, and other events. Although the SEFIR is critical for signaling, the molecular details of the functional subdomains within IL-17RA remain poorly characterized. Here, we provide a detailed structure-function analysis delineating the C-terminal boundary of the SEFIR-containing region of IL-17RA. We show that functionality of this domain requires a large extension to the previously identified SEFIR motif. In contrast to the SEFIR, this extension is not conserved among IL-17R family members. Surprisingly, Act1 recruitment is not sufficient for downstream signaling activation, whereas ubiquitination of TRAF6 correlates tightly with functional receptors. We further demonstrate that IL-17RA exhibits signaling properties that are nonredundant with other IL-17R family members. Finally, we report that IL-17 signals synergistically with lymphotoxin-α3, using the same signaling motifs within IL-17RA. These studies provide new insight into the structure-function relationships of IL-17RA and reveal distinct signaling differences among IL-17R family members.

Systemic administration of polymeric nanoparticle-encapsulated curcumin (NanoCurc) blocks tumor growth and metastases in preclinical models of pancreatic cancer.

Curcumin or diferuloylmethane is a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa). A large volume (several hundreds) of published reports has established the anticancer and chemopreventative properties of curcumin in preclinical models of every known major cancer type. Nevertheless, the clinical translation of curcumin has been significantly hampered due to its poor systemic bioavailability, which mandates that patients consume up to 8 to 10 g of the free drug orally each day to achieve detectable levels in circulation. We have engineered a polymeric nanoparticle encapsulated curcumin formulation (NanoCurc) that shows remarkably higher systemic bioavailability in plasma and tissues compared with free curcumin upon parenteral administration. In xenograft models of human pancreatic cancer established in athymic mice, administration of parenteral NanoCurc significantly inhibits primary tumor growth in both subcutaneous and orthotopic settings. The combination of parenteral NanoCurc with gemcitabine results in enhanced tumor growth inhibition versus either single agent, suggesting an additive therapeutic influence in vivo. Furthermore, this combination completely abrogates systemic metastases in orthotopic pancreatic cancer xenograft models. Tumor growth inhibition is accompanied by significant reduction in activation of nuclear factor-kappaB, as well as significant reduction in expression of matrix metalloproteinase-9 and cyclin D1, in xenografts treated with NanoCurc and gemcitabine. NanoCurc is a promising new formulation that is able to overcome a major impediment for the clinical translation of curcumin to cancer patients by improving systemic bioavailability, and by extension, therapeutic efficacy.

Magnesium phosphate nanoparticles can be efficiently used in vitro and in vivo as non-viral vectors for targeted gene delivery.

Magnesium phosphate (MgPi) nanoparticles (NPs) encapsulating pSVbetagal and pEGFP have been used as novel non-viral vector for targeted gene delivery. These plasmid DNA loaded magnesium phosphate nanoparticles of diameter 100-130 nm were prepared in water-in-oil microemulsion. In vitro cell viability study carried out on MCF-7, HEK, and COS-7 cells demonstrated that magnesium phosphate nanoparticles have no cytotoxic effect against cell proliferation. In vivo cytotoxicity conducted on Swiss Albino mice indicated no cytotoxic effect 3 months after intraperitoneal administration of 600 mg of void magnesium phosphate nanoparticles per Kg of body weight one-time only. In vitro transfection in COS-7 cells demonstrated that magnesium phosphate nanoparticles showed approximately 100% efficiency when compared to commercial transfecting reagent Polyfect as well as the transfection efficiency of calcium phosphate (CaPi) nanoparticles recently reported. Moreover, to explore the possibility of targeting these nanoparticles to specific tissue, we have surface modified these particles by adsorbing highlyt adhesive polymer, polyacrylic acid (PAA), followed by conjugating the carboxylic groups of the polymer with p-amino-thio-beta-galactopyranoside (PAG) using a cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and used these particles to target to liver in vivo successfully and more efficiently.

Dextran-doxorubicin/chitosan nanoparticles for solid tumor therapy.

Chemotherapy is a major therapeutic approach for the treatment of localized and metastasized cancers. Whereas potent chemotherapeutic agents seem promising in the test tube, clinical trials often fail due to unfavorable pharmacokinetics, poor delivery, low local concentrations, and limited accumulation in the target cell. The pathophysiology of the tumor vasculature and stromal compartment presents a major obstacle to effective delivery of agents to solid tumors. Poor perfusion of the tumor, arterio-venous shunting, necrotic and hypoxic areas, as well as a high interstitial fluid pressure work against favorable drug uptake. Thus, targeted drug delivery using long-circulating particulate drug carriers such as hydrogels of controlled size (<100 nm diameter) holds immense potential to improve the treatment of cancer by selectively providing therapeutically effective drug concentrations at the tumor site [through enhanced permeability and retention (EPR) effect] while reducing undesirable side effects. This review focuses on the progress of targeted delivery of nanoparticulated anticancer drug such as doxorubicin chemically conjugated with dextran and encapsulated in chitosan nanoparticles to solid tumor with reduced side effect of drug. Regulated particle size and long circulation of these hydrogel nanoparticles in blood help them accumulate in tumor tissue through EPR effect as evident from the significant regression of the tumor volume. The cardiotoxicity of doxorubicin can be minimized by coupling the drug with dextran and encapsulating it in chitosan nanoparticles.

In vivo characterization of a polymeric nanoparticle platform with potential oral drug delivery capabilities.

Nanotechnology has enabled significant advances in the areas of cancer diagnosis and therapy. The field of drug delivery is a sterling example, with nanoparticles being increasingly used for generating therapeutic formulations of poorly water-soluble, yet potent anticancer drugs. Whereas a number of nanoparticle-drug combinations are at various stages of preclinical or clinical assessment, the overwhelming majorities of such systems are injectable formulations and are incapable of being partaken orally. The development of an oral nano-delivery system would have distinct advantages for cancer chemotherapy. We report the synthesis and physicochemical characterization of orally bioavailable polymeric nanoparticles composed of N-isopropylacrylamide, methylmethacrylate, and acrylic acid in the molar ratios of 60:20:20 (designated NMA622). Amphiphilic NMA622 nanoparticles show a size distribution of <100 nm (mean diameter of 80 +/- 34 nm) with low polydispersity and can readily encapsulate a number of poorly water-soluble drugs such as rapamycin within the hydrophobic core. No apparent systemic toxicities are observed in mice receiving as much as 500 mg/kg of the orally administered void NMA622 for 4 weeks. Using NMA622-encapsulated rapamycin ("nanorapamycin") as a prototype for oral nano-drug delivery, we show favorable in vivo pharmacokinetics and therapeutic efficacy in a xenograft model of human pancreatic cancer. Oral nanorapamycin leads to robust inhibition of the mammalian target of rapamycin pathway in pancreatic cancer xenografts, which is accompanied by significant growth inhibition (P < 0.01) compared with control tumors. These data indicate that NMA622 nanoparticles provide a suitable platform for oral delivery of water-insoluble drugs like rapamycin for cancer therapy.

Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor.

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

Distinct functional motifs within the IL-17 receptor regulate signal transduction and target gene expression.

IL-17 is the founding member of a novel family of proinflammatory cytokines that defines a new class of CD4+ effector T cells, termed "Th17." Mounting evidence suggests that IL-17 and Th17 cells cause pathology in autoimmunity, but little is known about mechanisms of IL-17RA signaling. IL-17 through its receptor (IL-17RA) activates genes typical of innate immune cytokines, such as TNFalpha and IL-1beta, despite minimal sequence similarity in their respective receptors. A previous bioinformatics study predicted a subdomain in IL-17-family receptors with homology to a Toll/IL-1R (TIR) domain, termed the "SEFIR domain." However, the SEFIR domain lacks motifs critical for bona fide TIR domains, and its functionality was never verified. Here, we used a reconstitution system in IL-17RA-null fibroblasts to map functional domains within IL-17RA. We demonstrate that the SEFIR domain mediates IL-17RA signaling independently of classic TIR adaptors, such as MyD88 and TRIF. Moreover, we identified a previously undescribed"TIR-like loop" (TILL) required for activation of NF-kappaB, MAPK, and up-regulation of C/EBPbeta and C/EBPdelta. Mutagenesis of the TILL domain revealed a site analogous to the LPS(d) mutation in TLR4, which renders mice insensitive to LPS. However, a putative salt bridge typically found in TIR domains appears to be dispensable. We further identified a C-terminal domain required for activation of C/EBPbeta and induction of a subset IL-17 target genes. This structure-function analysis of a IL-17 superfamily receptor reveals important differences in IL-17RA compared with IL-1/TLR receptors.

Polymeric nanoparticle-encapsulated curcumin ("nanocurcumin"): a novel strategy for human cancer therapy.

BACKGROUND: Curcumin, a yellow polyphenol extracted from the rhizome of turmeric (Curcuma longa), has potent anti-cancer properties as demonstrated in a plethora of human cancer cell line and animal carcinogenesis models. Nevertheless, widespread clinical application of this relatively efficacious agent in cancer and other diseases has been limited due to poor aqueous solubility, and consequently, minimal systemic bioavailability. Nanoparticle-based drug delivery approaches have the potential for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus circumventing the pitfalls of poor solubility. RESULTS: We have synthesized polymeric nanoparticle encapsulated formulation of curcumin - nanocurcumin - utilizing the micellar aggregates of cross-linked and random copolymers of N-isopropylacrylamide (NIPAAM), with N-vinyl-2-pyrrolidone (VP) and poly(ethyleneglycol)monoacrylate (PEG-A). Physico-chemical characterization of the polymeric nanoparticles by dynamic laser light scattering and transmission electron microscopy confirms a narrow size distribution in the 50 nm range. Nanocurcumin, unlike free curcumin, is readily dispersed in aqueous media. Nanocurcumin demonstrates comparable in vitro therapeutic efficacy to free curcumin against a panel of human pancreatic cancer cell lines, as assessed by cell viability and clonogenicity assays in soft agar. Further, nanocurcumin's mechanisms of action on pancreatic cancer cells mirror that of free curcumin, including induction of cellular apoptosis, blockade of nuclear factor kappa B (NFkappaB) activation, and downregulation of steady state levels of multiple pro-inflammatory cytokines (IL-6, IL-8, and TNFalpha). CONCLUSION: Nanocurcumin provides an opportunity to expand the clinical repertoire of this efficacious agent by enabling ready aqueous dispersion. Future studies utilizing nanocurcumin are warranted in pre-clinical in vivo models of cancer and other diseases that might benefit from the effects of curcumin.