Single-wall carbon nanotubes (SWNTs) have been widely touted as attractive candidates for use as fillers in composite materials due to their extremely high Young's modulus, stiffness, and flexibility. 1 Successful applications of such composite systems require well-dispersed nanotubes with good adhesion with the host matrix, which, unfortunately, is not easily realized. Processing is rendered difficult by poor solubility of SWNTs, and the exfoliation of nanotube bundles is a major challenge. Moreover, inherently weak nanotube-polymer interactions result in poor interfacial adhesion, which can lead to nanotube aggregation within the matrix. Although a variety of chemical routes have been investigated to achieve nanotube solubility, 2 most methods either shorten the nanotubes or induce excessive functionalities that disrupt the original structure of the tubes. Polymer grafting, to improve the nanotube-polymer interface, has mainly been achieved on acidtreated nanotubes, 3 which may result in partial destruction of the tubular framework.Here, we report the development of a novel approach to in situ composite synthesis by attachment of polystyrene (PS) chains to full-length pristine SWNTs without disrupting the original structure, based on an established anionic polymerization scheme. 4 The process requires no nanotube pretreatment and works well with asproduced SWNTs. Both debundling of SWNT ropes and polymer attachment were achieved in a single step, and well-defined composites with a homogeneous dispersion of nanotubes were obtained.SWNTs produced by the HiPCO process 5 were used without further purification, as purification procedures might introduce functionalities that hinder carbanion formation. Dried pristine SWNTs were dispersed by sonication in purified cyclohexane. secButyllithium in slight excess of a predetermined amount (to ensure the removal of protic impurities on the SWNT surface) was added to this dispersion and sonicated in a bath for an hour. A homogeneous light yellow solution was obtained to which styrene monomer was added and polymerized at 48°C for 2 h under sonication. Carbanions are introduced on the SWNT surface by treatment with the anionic initiator that serves to exfoliate the bundles and provide initiating sites for the polymerization of styrene ( Figure 1). The negatively charged nanotubes are separated from the bundles and stay in solution due to mutual electrostatic repulsion between individual tubes, which was confirmed by long-term solution homogeneity. When styrene is added, both free secbutyllithium and the nanotube carbanions initiate polymerization, resulting in an intimately mixed composite system. The polymerization was terminated using degassed n-butanol, and the composite was recovered by precipitation with methanol. The composites were soluble in organic solvents such as dimethyl formamide, chloroform, and tetrahydrofuran.Composites with matrix molecular weights ranging from 1600 to 100 000 g mol -1 and polydispersities of ∼1.02 (determined using size exclusion chromatog...

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR 2 ). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G q -coupled PAR 2 calcium signaling; but all of these proteinases can disarm PAR 2 , releasing the N-terminal TL sequence, thereby preventing G q -coupled PAR 2 signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR 2 -mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ␤-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR 2 , downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR 2 signaling by preventing/disarming the G q /calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR 2 signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1 cleavage in or WT mice that received WT bone marrow than in WT mice that received bone marrow. Intravital microscopy of the kidney in mice 5-6 days after UUO demonstrated the significant recruitment of GFP proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CXCR1 renal phagocytes. , macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1, as well as the formation of AIM2 ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1 levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPSmediated IL-1b secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1b production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPSinduced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.