TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity, and the lack of sensing and in vivo imaging capabilities hamper the translation of most nanocarriers into clinic. To address this, we utilize nitrogen, boron/ nitrogen, and sulfur-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms allowing for multicolor visible/ near-IR imaging and cancer-sensing. These GQDs are scalably produced in one-step synthesis from a single biocompatible glucosamine precursor, are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36 h in biological environments as verified by TEM imaging. Because of their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion. Their high-yield intrinsic fluorescence in blue/ green and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores, and offers the capabilities for in vivo near-IR fluorescence tracking. Additionally, nitrogen-and sulfur-doped GQDs exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for the deterministic, ratiometric spectral discrimination between cancerous (HeLa and MCF-7 cell) versus healthy (HEK-293 cell) environments with substantial intensity ratios of 1.6 to 8. These results suggest fully biocompatible GQDs developed in this work as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

Several novel protein kinase C (PKC) isozymes have been identified as substrates for caspase-3. We have previously shown that novel PKC⑀ is cleaved during apoptosis in MCF-7 cells that lack any functional caspase-3. In the present study, we show that in vitro-translated PKC⑀ is processed by human recombinant caspase-3, -7, and -9. Tumor necrosis factor-␣ (TNF) triggered processing of PKC⑀ to a 43-kDa carboxyl-terminal fragment, and cell-permeable caspase inhibitors prevented TNF-induced processing of PKC⑀ in MCF-7 cells. PKC⑀ was cleaved primarily at the SSPD2G site to generate two fragments with an approximate molecular mass of 43 kDa. It was also cleaved at the DDVD2C site to generate two fragments with molecular masses of 52 and 35 kDa. Treatment of MCF-7 cells with TNF resulted in the activation of PKC⑀, and mutation at the SSPD2G (D383A) site inhibited proteolytic activation of PKC⑀. Overexpression of wild-type but not dominant-negative PKC⑀ in MCF-7 cells delayed TNF-induced apoptosis, and mutation at the D383A site prevented antiapoptotic activity of PKC⑀. These results suggest that cleavage of PKC⑀ by caspase-7 at the SSPD2G site results in the activation of PKC⑀. Furthermore, activation of PKC⑀ was associated with its antiapoptotic function.

This work describes the formation of porous composite materials based on a combination of bioactive mesoporous silicon and bioerodible polymers such as poly-caprolactone (PCL). The fabrication of a range of composites prepared by both salt leaching and microemulsion techniques are discussed. Particular attention to the influence of Si content in the composite on in vitro calcification assays are assessed. For each system, cytotoxicity and cellular proliferation are explicitly evaluated through fibroblast cell culture assays.

Graphene oxide (GO), the most common derivative of graphene, is an exceptional nanomaterial that possesses multiple physical properties critical for biomedical applications. GO exhibits pH-dependent fluorescence emission in the visible/near-infrared, providing a possibility of molecular imaging and pH-sensing. It is also water soluble and has a substantial platform for functionalization, allowing for the delivery of multiple therapeutics. GO physical properties are modified to enhance cellular internalization, producing fluorescent nanoflakes with low (<15%) cytotoxicity at the imaging concentrations of 15 μg/mL. As a result, at lower flake sizes GO rapidly internalizes into HeLa cells with the following 70% fluorescence based clearance at 24 h, assessed by its characteristic emission in red/near-IR. pH-dependence of GO emission is utilized to provide the sensing of acidic extracellular environments of cancer cells. The results demonstrate diminishing green/red (550/630 nm) fluorescence intensity ratios for HeLa and MCF-7 cancer cells in comparison to HEK-293 healthy cells suggesting a potential use of GO as a non-invasive optical sensor for cancer microenvironments. The results of this work demonstrate the potential of GO as a novel multifunctional platform for therapeutic delivery, biological imaging and cancer sensing.