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.     

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.     

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.     

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

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Effect of Exogenous Reductant on Growth and Iron Mobilization from Ferrihydrite by the Pseudomonas mendocina ymp Strain

Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.     

Downregulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 by grape seed proanthocyanidin extract

Urokinase plasminogen activator (uPA) system, comprising of uPA, its receptor uPAR and inhibitor, type 1 plasminogen activator inhibitor (PAI-1), plays a vital role in various biological processes involving extracellular proteolysis, fibrinolysis, cell migration and proliferation. The timely occurence of these processes are essential for normal wound healing. This study examines the regulation of uPA and PAI-1 by a natural polyphenol-rich compound, grape seed extract (GSE). GSE is reported to have beneficial effects in promoting wound healing. Fibroblast cells exposed to different doses of GSE for 18 hours were processed for further studies such as ELISA, RT-PCR, western blotting, fibrinolytic assay, cell surface plasmin activity assay and in vitro wound healing assay. GSE treatment caused a significant downregulation of uPA and PAI-1 expression, both at the RNA and protein levels. ELISA also revealed a dose-dependent decrease in uPA and PAI-1 activities. Functional significance of the downregulation was evident in decreased fibrinolytic activity, concomittant with decreased cell-surface plasmin activity. In vitro wound healing studies showed that GSE also retarded the migration of cells towards the wounded region.