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Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.
Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems.
© 2018 Cold Spring Harbor Laboratory Press.
Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish, Drosophila, C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions found in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover.
OBJECTIVES - This trial evaluated the efficacy of acetaminophen in reducing oxidative injury, as measured by plasma F2-isoprostanes, in adult patients with severe sepsis and detectable plasma cell-free hemoglobin.
DESIGN - Single-center, randomized, double-blind, placebo-controlled phase II trial.
SETTING - Medical ICU in a tertiary, academic medical center.
PATIENTS - Critically ill patients 18 years old or older with severe sepsis and detectable plasma cell-free hemoglobin.
INTERVENTIONS - Patients were randomized 1:1 to enteral acetaminophen 1 g every 6 hours for 3 days (n = 18) or placebo (n = 22) with the same dosing schedule and duration.
MEASUREMENTS AND MAIN RESULTS - F2-Isoprostanes on study day 3, the primary outcome, did not differ between acetaminophen (30 pg/mL; interquartile range, 24-41) and placebo (36 pg/mL; interquartile range, 25-80; p = 0.35). However, F2-isoprostanes were significantly reduced on study day 2 in the acetaminophen group (24 pg/mL; interquartile range, 19-36) when compared with placebo (36 pg/mL; interquartile range, 23-55; p = 0.047). Creatinine on study day 3, a secondary outcome, was significantly lower in the acetaminophen group (1.0 mg/dL; interquartile range, 0.6-1.4) when compared with that in the placebo (1.3 mg/dL; interquartile range, 0.83-2.0; p = 0.039). There was no statistically significant difference in hospital mortality (acetaminophen 5.6% vs placebo 18.2%; p = 0.355) or adverse events (aspartate aminotransferase or alanine aminotransferase > 400; acetaminophen 9.5% vs placebo 4.3%; p = 0.599).
CONCLUSIONS - In adults with severe sepsis and detectable plasma cell-free hemoglobin, treatment with acetaminophen within 24 hours of ICU admission may reduce oxidative injury and improve renal function. Additional study is needed to confirm these findings and determine the effect of acetaminophen on patient-centered outcomes.
OBJECTIVE - To determine the association of circulating cell-free hemoglobin with poor clinical outcomes in patients with sepsis and to characterize the potential protective effects of acetaminophen, an inhibitor of hemoprotein-mediated oxidation.
DESIGN - Retrospective observational study.
PATIENTS - A total of 391 critically ill patients with sepsis in multiple ICUs in an academic tertiary care hospital.
INTERVENTIONS - None.
MEASUREMENTS AND MAIN RESULTS - Nonsurvivors had significantly higher plasma cell-free hemoglobin concentrations (median 20mg/dL, interquartile range 10-40) measured on enrollment compared to survivors (10mg/dL, interquartile range 10-30, p = 0.002). After controlling for potential confounders, patients with higher cell-free hemoglobin concentrations were significantly more likely to die in the hospital (odds ratio 1.078, 95% confidence interval 1.012-1.149, p = 0.02). In addition, receiving acetaminophen in the setting of increased cell-free hemoglobin was independently associated with a protective effect against death (odds ratio 0.48, 95% confidence interval 0.25-0.91, p = 0.026) and lower plasma concentrations of the lipid peroxidation product F2-isoprostanes (18.5 pg/mL, interquartile range 9-22.2) compared to no acetaminophen (42 pg/mL, interquartile range 29.7-86, p = 0.009).
CONCLUSIONS - In critically ill patients with sepsis, elevated concentrations of circulating cell-free hemoglobin are independently associated with an increased risk of death. Acetaminophen may exert a protective effect by reducing cell-free hemoglobin-induced oxidative injury.
N(2),3-Ethenoguanine (N(2),3-εG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2'-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2'-fluoro-N(2),3-ε-2'-deoxyarabinoguanosine to investigate the miscoding potential of N(2),3-εG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N(2),3-εG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N(2),3-εG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N(2),3-εG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N(2),3-εG:dCTP base pair, whereas only one appears to be present in the case of the N(2),3-εG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N(2),3-εG.
Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.
Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets.
Cell-free systems can be used to reconstitute complex actin or microtubule-based phenomena. For example, a number of extracts support actin-dependent propulsion of Listeria monocytogenes, whereas Xenopus laevis extracts support formation of a microtubule-based meiotic spindle. Working in vitro opens these complex processes to biochemical dissection. Here, we describe methods to view these in vitro preparations by thin-section electron microscopy.