The electrochemical aptamer-based (E-AB) sensing platform appears to be a convenient (rapid, single-step, and calibration-free) and modular approach to measure concentrations of specific molecules (irrespective of their chemical reactivity) directly in blood and even in situ in the living *

The ability to monitor drugs, metabolites, hormones, and other biomarkers in situ in the body would greatly advance both clinical practice and biomedical research. To this end, we are developing electrochemical aptamer-based (EAB) sensors, a platform technology able to perform real-time, in vivo monitoring of specific molecules irrespective of their chemical or enzymatic reactivity. An important obstacle to the deployment of EAB sensors in the challenging environments found in the living body is signal drift, whereby the sensor signal decreases over time. To date, we have demonstrated a number of approaches by which this drift can be corrected sufficiently well to achieve good measurement precision over multihour in vivo deployments. To achieve a much longer in vivo measurement duration, however, will likely require that we understand and address the sources of this effect. In response, here, we have systematically examined the mechanisms underlying the drift seen when EAB sensors and simpler, EAB-like devices are challenged in vitro at 37 °C in whole blood as a proxy for in vivo conditions. Our results demonstrate that electrochemically driven desorption of a self-assembled monolayer and fouling by blood components are the two primary sources of signal loss under these conditions, suggesting targeted approaches to remediating this degradation and thus improving the stability of EAB sensors and other, similar electrochemical biosensor technologies when deployed in the body.

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ lag ) and maximum growth rate (ν max ) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ lag ∕∂½urea and ∂ log ν max ∕∂½urea) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.I ntracellular aggregation of the homodimeric enzyme superoxide dismutase (SOD1) is a key pathological hallmark of amyotrophic lateral sclerosis (ALS). Despite this coupling with protein-aggregation disease, SOD1 stands out as a relatively soluble protein in vitro: Mutations that are destabilized to the extent that they fail to fold can be left for weeks in physiological buffer without aggregating. Studies of SOD1 aggregation need then to involve certain stimuli that prompts the process to occur (1). One such stimulus is mechanical agitation. Like many other proteins, SOD1 can be driven to form amyloid-like fibrils by shaking or stirring the sample (2-4). Although these experiments may at first seem artificial, they have come to play a key role in our understanding of the aggregation mechanism: Protein aggregation is a high-dimensional process and, as such, its predicted outcome relies on knowledge about all competing pathways (5). In the case of SOD1, this high dimensionality is underlined by the observation that the structures of the fibrillar aggregates are polymorphic and vary with mutation (6). Competing aggregation pathways are also indicated at macroscopic level where the morphology of the SOD1 aggregates can be changed from filamentous to branched protofibrillar or amorphous by replacing the mechanical agitation with incubation in organic solvent (7), addition of lipids (8), or allowing oxidative cross-linking of the protein's solvent accessible cysteines (9). Non-amyloid aggregates can also be...

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric ␤-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent ␤-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in ␤-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous radical scavenger that is observed to misfold and aggregate into small intracellular inclusions in the motor neurons of patients with both familial and sporadic amyotrophic lateral sclerosis (ALS) 2 (1-3). As with other protein misfolding diseases (e.g. Alzheimer disease, Parkinson disease, and the prion diseases), the structural events underlying the pathological aggregation (4, 5) and also the role of this aggregation in the neurodegeneration are still obscure (6). Native SOD1 is a thermodynamically stable (7), long lived (8, 9), and structurally robust (10) homodimer that coordinates one redox-active Cu 1ϩ/2ϩ ion and one structural Zn 2ϩ ion in each subunit (7,(11)(12)(13). The Cu 1ϩ/2ϩ ion is coordinated directly to the protein's immunoglobulin-like scaffold, whereas the neighboring Zn 2ϩ ion is coordinated more peripherally by the long loop IV (Fig. 1). Loop IV also stretches across the SOD1 surface to support the dimer interface, contains the conserved disulfide bond between Cys-57 and Cys-146, and forms together with loop VII the rigid encasement of the metallated active site (Fig. 1). Upon dissociation of the metal ions, however, the active-site loops loosen up and become more dynamic (14 -18), coupled to a radical loss of protein stability (7, 9, 18) and increased propensity to aggregate (19 -22). Consistently, several in vivo studies (23) and fo...