Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
Although oxidative stress has been thought to play a general role in the activation of NF-κB, the involvement of reactive oxygen species (ROS) in facilitating nuclear translocation of NF-κB in neutrophils has not been described. In addition, the mechanisms by which ROS modulate the transcriptional activity of NF-κB in response to Toll-like receptor 4 (TLR4)-dependent signaling are not well characterized. To examine these issues, oxidant-dependent signaling events downstream of TLR4 were investigated in neutrophils stimulated with LPS. Pretreatment of neutrophils with the antioxidants N-acetylcysteine or α-tocopherol prevented LPS-induced nuclear translocation of NF-κB. Antioxidant treatment of LPS-stimulated neutrophils also inhibited the production of proinflammatory cytokines (TNF-α, macrophage inflammatory protein-2, and IL-1β), as well as activation of the kinases IκB kinase α, IκB kinase β, p38, Akt, and extracellular receptor-activated kinases 1 and 2. The decrease in cytoplasmic levels of IκBα produced by exposure of neutrophils to LPS was prevented by N-acetylcysteine or α-tocopherol. Activation of IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 in response to LPS stimulation was inhibited by antioxidants. These results demonstrate that proximal events in TLR4 signaling, at or antecedent to IRAK-1 and IRAK-4 activation, are oxidant dependent and indicate that ROS can modulate NF-κB-dependent transcription through their involvement in early TLR4-mediated cellular responses.
High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.
We herein report on the large-scale synthesis of ultrathin Bi(2)Te(3) nanoplates and subsequent spark plasma sintering to fabricate n-type nanostructured bulk thermoelectric materials. Bi(2)Te(3) nanoplates were synthesized by the reaction between bismuth thiolate and tri-n-octylphosphine telluride in oleylamine. The thickness of the nanoplates was ~1 nm, which corresponds to a single layer in Bi(2)Te(3) crystals. Bi(2)Te(3) nanostructured bulk materials were prepared by sintering of surfactant-removed Bi(2)Te(3) nanoplates using spark plasma sintering. We found that the grain size and density were strongly dependent on the sintering temperature, and we investigated the effect of the sintering temperature on the thermoelectric properties of the Bi(2)Te(3) nanostructured bulk materials. The electrical conductivities increased with an increase in the sintering temperature, owing to the decreased interface density arising from the grain growth and densification. The Seebeck coefficients roughly decreased with an increase in the sintering temperature. Interestingly, the electron concentrations and mobilities strongly depended on the sintering temperature, suggesting the potential barrier scattering at interfaces and the doping effect of defects and organic residues. The thermal conductivities also increased with an increase in the sintering temperature because of grain growth and densification. The maximum thermoelectric figure-of-merit, ZT, is 0.62 at 400 K, which is one of the highest among the reported values of n-type nanostructured materials based on chemically synthesized nanoparticles. This increase in ZT shows the possibility of the preparation of highly efficient thermoelectric materials by chemical synthesis.
The therapeutic potential of human multipotent mesenchymal stromal cells, especially human adipose tissue-derived stem cells (hASC), is promising. However, there are concerns about the safety of infusion of hASC in human. Recently, we have experienced pulmonary embolism and infarct among family members who have taken multiple infusions of intravenous autologous hASC therapy. A 41-year-old man presented with chest pain for one month. Chest CT showed multiple pulmonary artery embolism and infarct at right lung. Serum D-dimer was 0.8 µg/mL (normal; 0-0.5 µg/mL). He had received intravenous autologous adipose tissue-derived stem cell therapy for cervical herniated intervertebral disc three times (one, two, and three months prior to the visit). His parents also received the same therapy five times and their chest CT also showed multiple pulmonary embolism. These cases represent artificial pulmonary embolisms and infarct after IV injection of hASC. Follow-up chest CT showed spontaneous resolution of lesions in all three patients.
IntroductionFever is frequently observed in critically ill patients. An independent association of fever with increased mortality has been observed in non-neurological critically ill patients with mixed febrile etiology. The association of fever and antipyretics with mortality, however, may be different between infective and non-infective illness.MethodsWe designed a prospective observational study to investigate the independent association of fever and the use of antipyretic treatments with mortality in critically ill patients with and without sepsis. We included 1,425 consecutive adult critically ill patients (without neurological injury) requiring > 48 hours intensive care admitted in 25 ICUs. We recorded four-hourly body temperature and all antipyretic treatments until ICU discharge or 28 days after ICU admission, whichever occurred first. For septic and non-septic patients, we separately assessed the association of maximum body temperature during ICU stay (MAXICU) and the use of antipyretic treatments with 28-day mortality.ResultsWe recorded body temperature 63,441 times. Antipyretic treatment was given 4,863 times to 737 patients (51.7%). We found that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) or acetaminophen independently increased 28-day mortality for septic patients (adjusted odds ratio: NSAIDs: 2.61, P = 0.028, acetaminophen: 2.05, P = 0.01), but not for non-septic patients (adjusted odds ratio: NSAIDs: 0.22, P = 0.15, acetaminophen: 0.58, P = 0.63). Application of physical cooling did not associate with mortality in either group. Relative to the reference range (MAXICU 36.5°C to 37.4°C), MAXICU ≥ 39.5°C increased risk of 28-day mortality in septic patients (adjusted odds ratio 8.14, P = 0.01), but not in non-septic patients (adjusted odds ratio 0.47, P = 0.11).ConclusionsIn non-septic patients, high fever (≥ 39.5°C) independently associated with mortality, without association of administration of NSAIDs or acetaminophen with mortality. In contrast, in septic patients, administration of NSAIDs or acetaminophen independently associated with 28-day mortality, without association of fever with mortality. These findings suggest that fever and antipyretics may have different biological or clinical or both implications for patients with and without sepsis.Trial registrationClinicalTrials.gov: NCT00940654
Atelectasis caused by smooth bronchial narrowing, surrounded by calcified or noncalcified lymph nodes, in elderly and nonsmoking women is a typical finding of anthracofibrosis. Calcified lymph nodes adjacent to the involved bronchi and multifocal involvement of bronchial narrowing may be helpful in differentiating this condition from lung cancer.
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