Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease.

Previous studies suggest that single-walled carbon nanotube (SWCNT) exposure causes pulmonary fibrosis. We investigated the contribution of epithelial-mesenchymal transition (EMT) during SWCNT-induced pulmonary fibrosis. C57BL6 female mice were intratracheally instilled with SWCNT at 80 μg/mouse for up to 56 days. SWCNT exposure caused pulmonary epithelial and mesenchymal injury, followed by granulomatous and fibrotic changes. Immunofluorescence staining demonstrated the increasing occurrence of epithelial-derived fibroblasts up to 42 days post-exposure. Flow cytometry analysis revealed that 42.60% of N-cadherin (N-cad)-positive fibroblasts were derived from pulmonary epithelial cells, and, in separate experiments, 30.68% of SPC positive cells were stained for N-cad at 42 days. These epithelial-derived fibroblasts were functional in collagen production. With the progression of fibrosis, there were increases in the number of hyperplastic epithelial cells stained positively for TGF-β/p-Smad2 or β-catenin. Therefore, EMT contributes significantly to fibroblast expansion. Aberrant activations of TGF-β/p-Smad2 and β-catenin are postulated to induce EMT during SWCNT-induced pathogenic fibrosis.

A zinc-based metal organic framework, Zn-MOF-74, which has a unique one-dimensional (1D) channel and nanoscale aperture size, was rapidly obtained in 10 min using a de novo mild water-based system at room temperature, which is an example of green and sustainable chemistry. First, catalase (CAT) enzyme was encapsulated into Zn-MOF-74 (denoted as CAT@Zn-MOF-74), and comparative assays of biocatalysis, size-selective protection, and framework-confined effects were investigated. Electron microscopy and powder X-ray diffraction were used for characterization, while electrophoresis and confocal microscopy confirmed the immobilization of CAT molecules inside the single hexagonal MOF crystals at loading of ∼15 wt %. Furthermore, the CAT@Zn-MOF-74 hybrid was exposed to a denaturing reagent (urea) and proteolytic conditions (proteinase K) to evaluate its efficacy. The encapsulated CAT maintained its catalytic activity in the decomposition of hydrogen peroxide (H 2 O 2 ), even when exposed to 0.05 M urea and proteinase K, yielding an apparent observed rate constant (k obs ) of 6.0 × 10 −2 and 6.6 × 10 −2 s −1 , respectively. In contrast, free CAT exhibited sharply decreased activity under these conditions. Additionally, the bioactivity of CAT@Zn-MOF-74 for H 2 O 2 decomposition was over three times better than that of the biocomposites based on zeolitic imidazolate framework 90 (ZIF-90) owing to the nanometer-scaled apertures, 1D channel, and less confinement effects in Zn-MOF-74 crystallites. To demonstrate the general applicability of this strategy, another enzyme, α-chymotrypsin (CHT), was also encapsulated in Zn-MOF-74 (denoted as CHT@Zn-MOF-74) for action against a substrate larger than H 2 O 2 . In particular, CHT@Zn-MOF-74 demonstrated a biological function in the hydrolysis of Lphenylalanine p-nitroanilide (HPNA), the activity of ZIF-90-encapsulated CHT was undetectable due to aperture size limitations. Thus, we not only present a rapid eco-friendly approach for Zn-MOF-74 synthesis but also demonstrate the broader feasibility of enzyme encapsulation in MOFs, which may help to meet the increasing demand for their industrial applications.

Extracellular deposits of amyloid β (Aβ) aggregates in the brain is the hallmark of Alzheimer's disease. We present the configurations (location and conformation) and the interfacial folding and membrane insertion mechanisms of Aβ fragments, wild-type Aβ(25-35), Aβ(35-25), and a sequence-shuffled peptide [Aβ(25-35)-shuffled] from Aβ(25-35) within membranes by replica-exchange molecular dynamics simulations. Although these peptides have the same amino acid composition, simulations show they have distinct locations and conformations within membranes. Moreover, our in vitro experiments show that these peptides have distinct neurotoxicities. We rationalize the distinct neurotoxicities of these peptides in terms of their simulated locations and conformations in membranes. This work provides another view that complements the general hydrophobicity-toxicity views, to better explain the neurotoxicity of Aβ peptides.