Characterization and structure of a Zn2+ and [2Fe-2S]-containing copper chaperone from Archaeoglobus fulgidus

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.     

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

Regenerating Corticospinal Axons Innervate Phenotypically Appropriate Neurons within Neural Stem Cell Grafts

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Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen''s homolog of the mitochondrial bc₁ complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a “supercomplex” with a cytochrome aa₃-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa₃ supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.     

Streptococcus sanguinis Class Ib Ribonucleotide Reductase: HIGH ACTIVITY WITH BOTH IRON AND MANGANESE COFACTORS AND STRUCTURAL INSIGHTS*

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe^II and O₂ can self-assemble a diferric-tyrosyl radical (Fe^III₂-Y^•) cofactor (1.2 Y^•/β₂) and with the help of NrdI can assemble a Mn^III₂-Y^• cofactor (0.9 Y^•/β₂). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdI_ox and Mn^II₂-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.     

Carbon–Temperature–Water change analysis for peanut production under climate change: a prototype for the AgMIP Coordinated Climate‐Crop Modeling Project (C3MP)

Climate change is projected to push the limits of cropping systems and has the potential to disrupt the agricultural sector from local to global scales. This article introduces the Coordinated Climate‐Crop Modeling Project (C3MP), an initiative of the Agricultural Model Intercomparison and Improvement Project (AgMIP) to engage a global network of crop modelers to explore the impacts of climate change via an investigation of crop responses to changes in carbon dioxide concentration ([CO₂]), temperature, and water. As a demonstration of the C3MP protocols and enabled analyses, we apply the Decision Support System for Agrotechnology Transfer (DSSAT) CROPGRO‐Peanut crop model for Henry County, Alabama, to evaluate responses to the range of plausible [CO₂], temperature changes, and precipitation changes projected by climate models out to the end of the 21st century. These sensitivity tests are used to derive crop model emulators that estimate changes in mean yield and the coefficient of variation for seasonal yields across a broad range of climate conditions, reproducing mean yields from sensitivity test simulations with deviations of ca. 2% for rain‐fed conditions. We apply these statistical emulators to investigate how peanuts respond to projections from various global climate models, time periods, and emissions scenarios, finding a robust projection of modest (<10%) median yield losses in the middle of the 21st century accelerating to more severe (>20%) losses and larger uncertainty at the end of the century under the more severe representative concentration pathway (RCP8.5). This projection is not substantially altered by the selection of the AgMERRA global gridded climate dataset rather than the local historical observations, differences between the Third and Fifth Coupled Model Intercomparison Project (CMIP3 and CMIP5), or the use of the delta method of climate impacts analysis rather than the C3MP impacts response surface and emulator approach.     

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction

Introduction

Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.

Methods

Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.

Results

In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.

Conclusions

These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0419-y) contains supplementary material, which is available to authorized users.     

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.