Aging aggravates ischemic stroke‐induced brain damage in mice with chronic peripheral infection

Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all of which alter peripheral inflammatory processes with concomitant impact on stroke outcome. The majority of the stroke patients are elderly, but the impact of interactions between aging and inflammation on stroke remains unknown. We thus investigated the influence of age on the outcome of stroke in animals predisposed to systemic chronic infection. Th1‐polarized chronic systemic infection was induced in 18–22 month and 4‐month‐old C57BL/6j mice by administration of Trichuris muris (gut parasite). One month after infection, mice underwent permanent middle cerebral artery occlusion and infarct size, brain gliosis, and brain and plasma cytokine profiles were analyzed. Chronic infection increased the infarct size in aged but not in young mice at 24 h. Aged, ischemic mice showed altered plasma and brain cytokine responses, while the lesion size correlated with plasma prestroke levels of RANTES. Moreover, the old, infected mice exhibited significantly increased neutrophil recruitment and upregulation of both plasma interleukin‐17α and tumor necrosis factor‐α levels. Neither age nor infection status alone or in combination altered the ischemia‐induced brain microgliosis. Our results show that chronic peripheral infection in aged animals renders the brain more vulnerable to ischemic insults, possibly by increasing the invasion of neutrophils and altering the inflammation status in the blood and brain. Understanding the interactions between age and infections is crucial for developing a better therapeutic regimen for ischemic stroke and when modeling it as a disease of the elderly.     

Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.     

Crystal structure of ferrioxamine B: a comparative analysis and implications for molecular recognition

Ferrioxamine B was successfully co-crystallized with ethanolpentaaquomagnesium(II) and perchlorate ions as counter ions, C27H62Cl3FeMgN6O26, and the crystal structure has been determined by single-crystal X-ray diffraction. The crystals are monoclinic, space group P2(1)/n, four molecules per unit cell with dimensions a=21.1945(7) A, b=10.0034(3) A, c=106.560(1) A, and beta=106.560(1) degrees. The crystal structure contains a racemic mixture of Lambda-N-cis,cis and Delta-N-cis,cis coordination isomers. The structural parameters and the conformational features of ferrioxamine B compare very well with those of ferrioxamines D1 and E, with an exception of the orientation of the pendant protonated amine, which is pointing away from the connecting amide chains and towards the carbonyl face of the inner coordination shell distorted octahedron. This pendant protonated amine, in conjunction with the carbonyl face of the Fe(III) coordination shell, is proposed to play an important role in the recognition and membrane transport processes.     

Leopard (Panthera pardus) occupancy in the Chure range of Nepal

Conservation of large carnivores such as leopards requires large and interconnected habitats. Despite the wide geographic range of the leopard globally, only 17% of their habitat is within protected areas. Leopards are widely distributed in Nepal, but their population status and occupancy are poorly understood. We carried out the sign‐based leopard occupancy survey across the entire Chure range (~19,000 km²) to understand the habitat occupancy along with the covariates affecting their occupancy. Leopard signs were obtained from in 70 out of 223 grids surveyed, with a naïve leopard occupancy of 0.31. The model‐averaged leopard occupancy was estimated to be 0.5732 (SE 0.0082) with a replication‐level detection probability of 0.2554 (SE 0.1142). The top model shows the additive effect of wild boar, ruggedness, presence of livestock, and human population density positively affecting the leopard occupancy. The detection probability of leopard was higher outside the protected areas, less in the high NDVI (normalized difference vegetation index) areas, and higher in the areas with livestock presence. The presence of wild boar was strong predictor of leopard occupancy followed by the presence of livestock, ruggedness, and human population density. Leopard occupancy was higher in west Chure (0.70 ± SE 0.047) having five protected areas compared with east Chure (0.46 ± SE 0.043) with no protected areas. Protected areas and prey species had positive influence on leopard occupancy in west Chure range. Similarly in the east Chure, the leopard occupancy increased with prey, NDVI, and terrain ruggedness. Enhanced law enforcement and mass awareness activities are necessary to reduce poaching/killing of wild ungulates and leopards in the Chure range to increase leopard occupancy. In addition, maintaining the sufficient natural prey base can contribute to minimize the livestock depredation and hence decrease the human–leopard conflict in the Chure range.     

Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3

S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.     

Yale school of public health symposium on lifetime exposures and human health: the exposome; summary and future reflections

The exposome is defined as “the totality of environmental exposures encountered from birth to death” and was developed to address the need for comprehensive environmental exposure assessment to better understand disease etiology. Due to the complexity of the exposome, significant efforts have been made to develop technologies for longitudinal, internal and external exposure monitoring, and bioinformatics to integrate and analyze datasets generated. Our objectives were to bring together leaders in the field of exposomics, at a recent Symposium on “Lifetime Exposures and Human Health: The Exposome,” held at Yale School of Public Health. Our aim was to highlight the most recent technological advancements for measurement of the exposome, bioinformatics development, current limitations, and future needs in environmental health. In the discussions, an emphasis was placed on moving away from a one-chemical one-health outcome model toward a new paradigm of monitoring the totality of exposures that individuals may experience over their lifetime. This is critical to better understand the underlying biological impact on human health, particularly during windows of susceptibility. Recent advancements in metabolomics and bioinformatics are driving the field forward in biomonitoring and understanding the biological impact, and the technological and logistical challenges involved in the analyses were highlighted. In conclusion, further developments and support are needed for large-scale biomonitoring and management of big data, standardization for exposure and data analyses, bioinformatics tools for co-exposure or mixture analyses, and methods for data sharing.     

In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions

1. Download : Download high-res image (247KB)
2. Download : Download full-size image

     

SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) analysis of the thermodynamics of synergistic anion binding by ferric-binding protein (FbpA), a bacterial transferrin

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA. As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)). The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.