Separation of proteins by cation-exchange sequential injection chromatography using a polymeric monolithic column

Abstract

Since sequential injection chromatography (SIC) emerged in 2003, it has been used for separation of small molecules in diverse samples, but separations of high molar mass compounds such as proteins have not yet been described. In the present work a poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic column was prepared by free radical polymerization inside a 2.1-mm-i.d. activated fused silica-lined stainless steel tubing and modified with iminodiacetic acid (IDA). The column was prepared from a mixture of 24 % GMA, 16 % EDMA, 20 % cyclohexanol, and 40 % 1-dodecanol (all % as w/w) containing 1 % of azobisisobutyronitrile (AIBN) (in relation to monomers). Polymerization was done at 60 °C for 24 h. The polymer was modified with 1.0 M IDA (in 2 M Na₂CO₃, pH 10.5) at 80 °C for 16 h. Separation of myoglobin, ribonuclease A, cytochrome C, and lysozyme was achieved at pH 7.0 (20 mM KH₂PO₄/K₂HPO₄) using a salt gradient (NaCl). Myoglobin was not retained, and the other proteins were separated by a gradient of NaCl created inside the holding coil (4 m of 0.8-mm-i.d. PTFE tubing) by sequential aspiration of 750 and 700 μL of 0.2 and 0.1 M NaCl, respectively. As the flow was reversed toward the column (5 μL s⁻¹) the interdispersion of these solutions created a reproducible gradient which separated the proteins in 10 min, with the following order of retention: ribonuclease A < cytochrome C < lysozyme. The elution order was consistent with a cation-exchange mechanism as the retention increased with the isoelectric points.

Graphical Abstract

Sequential injection chromatograph for cation-exchange separation of proteins in a iminodiacetatemodified polyglycidyl methacrylate-co-ethylene dimethacrylate, IDA-poly(GMA-co-EDMA), monolithic column, where MP₁ = 20 mM KH₂PO₄/K₂HPO₄ (pH 7.0), HC = Holding coil, SP = Syringe Pump, RV = Relef Valve and D = UV-Detector

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PAL SPME Arrow—evaluation of a novel solid-phase microextraction device for freely dissolved PAHs in water

Abstract

After more than 25 years, solid-phase microextraction (SPME) has gained widespread acceptance as a well-automatable and flexible microextraction technique, while its instrumental basis remained mostly unchanged. The novel PAL (Prep And Load solution) SPME Arrow combines the advantages of SPME with the benefits of extraction techniques providing larger sorption phase volumes such as stir bar sorptive extraction (SBSE). It thereby avoids the inherent drawbacks of both techniques such as limitations in method automation in the case of SBSE, as well as the small sorption phase volumes and the lacking fiber robustness of classical SPME fibers. This new design is based on a robust stainless steel backbone, carrying, the screw connection to the PAL sampler, the enlarged sorption phase, and an arrow-shaped tip for conservative penetration of septa (hence the name). An outer capillary encloses this phase apart from enrichment and desorption processes and rests against the tip during transfer and penetrations, resulting in a homogeneously closed device. Here, we present an evaluation and a comparison of the novel PAL SPME Arrow with classical SPME fibers, extracting polycyclic aromatic hydrocarbons (PAHs) as model analytes, from the freely dissolved fraction in lab water and groundwater via direct immersion using polydimethylsiloxane (PDMS) as common sorption phase material. Limits of detection, repeatabilities, and extraction yields were determined for the PAL SPME Arrow and compared to data of classical SPME fibers and SBSE bars. Results indicate a significant benefit in extraction efficiency due to the larger sorption phase volume. It is accompanied by faultless mechanical robustness and thus better reliability, especially in case of prolonged, unattended, and automated operation. As an exemplary application, the water-soluble fraction of PAHs and derivatives in a roofing felt sample was quantified.

Graphical Abstract

Picture of a PAL SPME Arrow during extraction of a stirred water sample

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Optimization and comparison of miniaturized extraction techniques for PAHs from crude oil exposed Atlantic cod and haddock eggs

Abstract

Two miniaturized extraction methods for a wide range of 2-6 ring polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in small lipid-rich biota samples (≤100 mg) have been developed. Both methods utilize liquid extraction (LE) prior to a clean-up step using either normal phase solid phase extraction (SPE) or mixed-phase dispersive SPE (dSPE). Optimization of the methods was achieved by comparing the type and amount of sorbents, drying agents, and solvents used. In order to improve the limits of detection (LOD) of target PAHs under high sensitivity gas chromatography–tandem mass spectrometry analysis, specific emphasis was given to minimizing lipid co-extraction. The optimized LE–SPE method comprised extraction with dichloromethane/n-hexane (1:1, v/v) and clean-up by silica SPE, whereas the optimized LE–dSPE method comprised extraction with acetonitrile and clean-up with PSA and C18 sorbents. The methods were validated and directly compared through the analysis of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) eggs exposed to oil. The LE–SPE method resulted in lower levels of co-extracted lipids (14.1 ± 1.7 ng/μL) than the LE–dSPE method (60 ± 14 ng/μL). Achieved PAH LODs for the LE–SPE method were typically an order of magnitude lower (<5 ng/g) than for the LE–dSPE method (<125 ng/g). The LE–SPE method offers the possibility for PAH analysis of small samples of fish eggs (~100 mg) exposed to small quantities of crude oil (~1–10 μg/L total PAHs).

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Development of CT-guided biopsy sampling for time-dependent postmortem redistribution investigations in blood and alternative matrices—proof of concept and application on two cases

Abstract

The postmortem redistribution (PMR) phenomenon complicates interpretation in forensic toxicology. Human data on time-dependent PMR are rare and only exist for blood so far. A new method for investigation of time-dependent PMR in blood as well as in alternative body fluids and tissues was developed and evaluated using automated biopsy sampling. At admission of the bodies, introducer needles were placed in liver, lung, kidney, muscle, spleen, adipose tissue, heart, femoral vein, and lumbar spine using a robotic arm guided by a computed tomography scanner (CT). Needle placement accuracy was analyzed and found to be acceptable for the study purpose. Tissue biopsies and small volume body fluid samples were collected in triplicate through the introducer needles. At autopsy (around 24 h after admission), samples from the same body regions were collected. After mastering of the technical challenges, two authentic cases were analyzed as a proof of concept. Drug concentrations of venlafaxine, O-desmethylvenlafaxine, bromazepam, flupentixol, paroxetine, and lorazepam were determined by LC-MS/MS, and the percentage concentration changes between the two time points were calculated. Concentration changes were observed with both increases and decreases depending on analyte and matrix. While venlafaxine, flupentixol, paroxetine, and lorazepam generally showed changes above 30 % and more, O-desmethylvenlafaxine and bromazepam did not undergo extensive PMR. The presented study shows that CT-controlled biopsy collection provides a valuable tool for systematic time-dependent PMR investigation, demanding only minimal sample amount and causing minimal damage to the body.

Graphical abstract

New strategy for time-dependent postmortem redistribution studies by CT guided biopsy sampling prior to autopsy and comparison of drug concentrations at these two time points using validated LC-MS/MS quantitation.

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Comprehensive analysis of serum metabolites in gestational diabetes mellitus by UPLC/Q-TOF-MS

Abstract

Gestational diabetes mellitus (GDM) refers to the first sign or onset of diabetes mellitus during pregnancy rather than progestation. In recent decades, more and more research has focused on the etiology and pathogenesis of GDM in order to further understand GDM progress and recovery. Using an advanced metabolomics platform based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS), we explored the changes in serum metabolites between women with GDM and healthy controls during and after pregnancy. Some significant differences were discovered using multivariate analysis including partial least-squares discriminant analysis (PLS-DA) and orthogonal PLS-DA (OPLS-DA). The dysregulated metabolites were further compared and verified in several databases to understand how these compounds might function as potential biomarkers. Analyses of the metabolic pathways associated with these potential biomarkers were subsequently explored. A total of 35 metabolites were identified, contributing to GDM progress to some extent. The identified biomarkers were involved in some important metabolic pathways including glycine, serine, and threonine metabolism; steroid hormone biosynthesis; tyrosine metabolism; glycerophospholipid metabolism; and fatty acid metabolism. The above mentioned metabolic pathways mainly participate in three major metabolic cycles in humans, including lipid metabolism, carbohydrate metabolism, and amino acid metabolism. In this pilot study, the valuable comprehensive analysis gave us further insight into the etiology and pathophysiology of GDM, which might benefit the feasibility of a rapid, accurate diagnosis and reasonable treatment as soon as possible but also prevent GDM and its related short- and long-term complications.

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Detection of volatile organic compounds (VOCs) from exhaled breath as noninvasive methods for cancer diagnosis

Abstract

The detection of cancer at an early stage is often significant in the successful treatment of the disease. Tumor cells have been reported to generate unique cancer volatile organic compound (VOC) profiles which can reflect the disease conditions. The detection and analysis of VOC biomarkers from exhaled breath has been recognized as a new frontier in cancer diagnostics and health inspections owing to its potential in developing rapid, noninvasive, and inexpensive cancer screening tools. To detect specific VOCs of low concentrations from exhaled breath, and to enhance the accuracy of early diagnosis, many breath collection and analysis approaches have been developed. This paper will summarize and critically review the exhaled-breath VOC-related sampling, collection, detection, and analytical methods, especially the recent development in VOC sensors. VOC sensors are commonly inexpensive, portable, programmable, easy to use, and can obtain data in real time with high sensitivities. Therefore, many sensor-based VOC detection techniques have huge potential in clinical point-of-care use.

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Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports

Abstract

The detection of testosterone abuse in sports is routinely achieved through the 'steroidal module' of the Athlete Biological Passport by GC-MS(/MS) quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes' urines. To overcome some limitations of the "urinary steroid profile" such as the presence of confounding factors (ethnicity, enzyme polymorphism, bacterial contamination, and ethanol), ultrahigh performance liquid chromatography (UHPLC) measurements of blood concentrations of testosterone, its major metabolites, and precursors could represent an interesting and complementary strategy. In this work, two UHPLC-MS/MS methods were developed for the quantification of testosterone and related compounds in human serum, including major progestogens, corticoids, and estrogens. The validated methods were then used for the analyses of serum samples collected from 19 healthy male volunteers after oral and transdermal testosterone administration. Results from unsupervised multiway analysis allowed variations of target analytes to be assessed simultaneously over a 96-h time period. Except for alteration of concentration values due to the circadian rhythm, which concerns mainly corticosteroids, DHEA, and progesterone, significant variations linked to the oral and transdermal testosterone administration were observed for testosterone, DHT, and androstenedione. As a second step of analysis, the longitudinal monitoring of these biomarkers using intra-individual thresholds showed, in comparison to urine, significant improvements in the detection of testosterone administration, especially for volunteers with del/del genotype for phase II UGT2B17 enzyme, not sensitive to the main urinary marker, T/E ratio. A substantial extension of the detection window after transdermal testosterone administration was also observed in serum matrix. The longitudinal follow-up proposed in this study represents a first example of 'blood steroid profile' in doping control analysis, which can be proposed in the future as a complement to the 'urinary module' for improving steroid abuse detection capabilities.

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Surface-enhanced Raman spectroscopy in 3D electrospun nanofiber mats coated with gold nanorods

Abstract

Nanofibers functionalized by metal nanostructures and particles are exploited as effective flexible substrates for surface-enhanced Raman scattering (SERS) analysis. Their complex three-dimensional structure may provide Raman signals enhanced by orders of magnitude compared to untextured surfaces. Understanding the origin of such improved performances is therefore very important for pushing nanofiber-based analytical technologies to their upper limit. Here, we report on polymer nanofiber mats which can be exploited as substrates for enhancing the Raman spectra of adsorbed probe molecules. The increased surface area and the scattering of light in the nanofibrous system are individually analyzed as mechanisms to enhance Raman scattering. The deposition of gold nanorods on the fibers further amplifies Raman signals due to SERS. This study suggests that Raman signals can be finely tuned in intensity and effectively enhanced in nanofiber mats and arrays by properly tailoring the architecture, composition, and light-scattering properties of the complex networks of filaments.

Graphical abstract

Enhancement of Raman signals by electrospun nanofiber mats

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Comparative study of the oxidation behavior of sulfur-containing amino acids and glutathione by electrochemistry-mass spectrometry in the presence and absence of cisplatin

Abstract

Small sulfur-containing compounds are involved in several important biochemical processes, including—but not limited to—redox regulation and drug conjugation/detoxification. While methods for stable redox pairs of such compounds (thiols/disulfides) are available, analytical data on more labile and short-lived redox intermediates are scarce, due to highly challenging analytical requirements. In this study, we employ the direct combination of reagentless electrochemical oxidation and mass spectrometric (EC-MS) identification for monitoring oxidation reactions of cysteine, N-acetylcysteine, methionine, and glutathione under simulated physiological conditions (pH 7.4, 37 °C). For the first time, all theoretically expected redox intermediates—with only one exception—are detected simultaneously and in situ, including sulfenic, sulfinic, and sulfonic acids, disulfides, thiosulfinates, thiosulfonates, and sulfoxides. By monitoring the time/potential-dependent interconversion of sulfur species, mechanistic oxidation routes are confirmed and new reactions detected, e.g., sulfenamide formation due to reaction with ammonia from the buffer. Furthermore, our results demonstrate a highly significant impact of cisplatin on the redox reactivity of sulfur species. Namely, the amount of thiol oxidation to sulfonic acid via sulfenic and sulfinic acid intermediates is diminished for glutathione in the presence of cisplatin in favor of the disulfide formation, while for N-acetylcysteine the contrary applies. N-acetylcysteine is the only ligand which displays enhanced oxidation currents upon cisplatin addition, accompanied by increased levels of thiosulfinate and thiosulfonate species. This is traced back to thiol reactivity and highlights the important role of sulfenic acid intermediates, which may function as a switch between different oxidation routes.

Graphical Abstract

Electrochemistry-Mass spectrometry reveals changes in the oxidation pathway of thiols in the presence of cisplatin and enables detection of labile reaction intermediates

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Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis

Abstract

Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time <60 s) and method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid–liquid extraction (SALLE) with volatile solutes and NH₄⁺ QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits 'in the middle' between ambient MS and chromatography-MS, offering a balance between analytical capability and sample analysis throughput suitable for broad applications in life sciences, agricultural chemistry, consumer safety, and beyond.

Graphical abstract

Position of FIA-MS relative to chromatography-MS and ambient MS in terms of analytical figures of merit and sample analysis throughput.

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SpinCouple: Development of a Web Tool for Analyzing Metabolite Mixtures via Two-Dimensional J-Resolved NMR Database

Analytical Chemistry

DOI: 10.1021/acs.analchem.5b02311

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Fentanyl Pharmacokinetics in Critically Ill Patients: A Demonstration of Mixed Effects*

No abstract available

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Equity of Access to Critical Care Among Elderly Patients in Scotland: A National Cohort Study*

Objective: To compare elderly (≥ 80 yr), older (65–79 yr), and younger (

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Ventilator-Associated Events in Neonates and Children—A New Paradigm*

Objectives: To identify a pediatric ventilator-associated condition definition for use in neonates and children by exploring whether potential ventilator-associated condition definitions identify patients with worse outcomes. Design: Retrospective cohort study and a matched cohort analysis. Setting: Pediatric, cardiac, and neonatal ICUs in five U.S. hospitals. Patients: Children 18 years old or younger ventilated for at least 1 day. Interventions: None. Measurements and Main Results: We evaluated the evidence of worsening oxygenation via a range of thresholds for increases in daily minimum fraction of inspired oxygen (by 0.20, 0.25, and 0.30) and daily minimum mean airway pressure (by 4, 5, 6, and 7 cm H2O). We required worsening oxygenation be sustained for at least 2 days after at least 2 days of stability. We matched patients with a ventilator-associated condition to those without and used Cox proportional hazard models with frailties to examine associations with hospital mortality, hospital and ICU length of stay, and duration of ventilation. The cohort included 8,862 children with 10,209 hospitalizations and 77,751 ventilator days. For the fraction of inspired oxygen 0.25/mean airway pressure 4 definition (i.e., increase in minimum daily fraction of inspired oxygen by 0.25 or mean airway pressure by 4), rates ranged from 2.9 to 3.2 per 1,000 ventilator days depending on ICU type; the fraction of inspired oxygen 0.30/mean airway pressure 7 definition yielded ventilator-associated condition rates of 1.1–1.3 per 1,000 ventilator days. All definitions were significantly associated with greater risk of hospital death, with hazard ratios ranging from 1.6 (95% CI, 0.7–3.4) to 6.8 (2.9–16.0), depending on thresholds and ICU type. Each definition was associated with prolonged hospitalization, time in ICU, and duration of ventilation, among survivors. The advisory board of the study proposed using the fraction of inspired oxygen 0.25/mean airway pressure 4 thresholds to identify pediatric ventilator-associated conditions in ICUs. Conclusions: Pediatric patients with ventilator-associated conditions are at substantially higher risk for mortality and morbidity across ICUs, regardless of thresholds used. Next steps include identification of risk factors, etiologies, and preventative measures for pediatric ventilator-associated conditions.

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Hyperbaric Oxygen Therapy Alleviates Carbon Monoxide Poisoning–Induced Delayed Memory Impairment by Preserving Brain-Derived Neurotrophic Factor–Dependent Hippocampal Neurogenesis

Objective: To test the hypothesis that hyperbaric oxygen therapy ameliorates delayed cognitive impairment after acute carbon monoxide poisoning by promoting neurogenesis through upregulating the brain-derived neurotrophic factor in the hippocampus. Design: Laboratory animal experiments. Setting: University/Medical center research laboratory. Subjects: Adult, male Sprague-Dawley rats. Interventions: Rats were divided into five groups: (1) non–carbon monoxide-treated control, (2) acute carbon monoxide poisoning, (3) acute carbon monoxide poisoning followed by 7-day hyperbaric oxygen treatment, (4) carbon monoxide + hyperbaric oxygen with additional intracerebroventricular infusion of Fc fragment of tyrosine kinase receptor B protein (TrkB-Fc) chimera, and (5) acute carbon monoxide poisoning followed by intracerebroventricular infusion of brain-derived neurotrophic factor. Acute carbon monoxide poisoning was achieved by exposing the rats to carbon monoxide at 2,500 ppm for 40 minutes, followed by 3,000 ppm for 20 minutes. Hyperbaric oxygen therapy (at 2.5 atmospheres absolute with 100% oxygen for 60 min) was conducted during the first 7 days after carbon monoxide poisoning. Recombinant human TrkB-Fc chimera or brain-derived neurotrophic factor was infused into the lateral ventricle via the implanted osmotic minipump. For labeling of mitotic cells in the hippocampus, bromodeoxyuridine was injected into the peritoneal cavity. Distribution of bromodeoxyuridine and two additional adult neurogenesis markers, Ki-67 and doublecortin, in the hippocampus was evaluated by immunohistochemistry or immunofluorescence staining. Tissue level of brain-derived neurotrophic factor was assessed by enzyme-linked immunosorbent assay. Cognitive behavior was evaluated by the use of eight-arm radial maze. Measurements and Main Results: Acute carbon monoxide poisoning significantly suppressed adult hippocampal neurogenesis evident by the reduction in number of bromodeoxyuridine-positive, Ki-67+, and doublecortin+ cells in the subgranular zone of the dentate gyrus. This suppression of adult neurogenesis by the carbon monoxide poisoning was appreciably alleviated by early treatment of hyperbaric oxygen. The hyperbaric oxygen treatment also promoted a sustained increase in hippocampal brain-derived neurotrophic factor level. Blockade of hippocampal brain-derived neurotrophic factor signaling with intracerebroventricular infusion of recombinant human TrkB-Fc chimera significantly blunted the protection by the hyperbaric oxygen on hippocampal neurogenesis; whereas intracerebroventricular infusion of brain-derived neurotrophic factor mimicked the action of hyperbaric oxygen and preserved hippocampal neurogenesis after acute carbon monoxide poisoning. Furthermore, acute carbon monoxide poisoning resulted in a delayed impairment of cognitive function. The hyperbaric oxygen treatment notably restored the cognitive impairment in a brain-derived neurotrophic factor–dependent manner. Conclusions: The early hyperbaric oxygen treatment may alleviate delayed memory impairment after acute carbon monoxide poisoning by preserving adult neurogenesis via an increase in hippocampal brain-derived neurotrophic factor content.

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A Selective V1A Receptor Agonist, Selepressin, Is Superior to Arginine Vasopressin and to Norepinephrine in Ovine Septic Shock*

Objective: Selective vasopressin V1A receptor agonists may have advantages over arginine vasopressin in the treatment of septic shock. We compared the effects of selepressin, a selective V1A receptor agonist, arginine vasopressin, and norepinephrine on hemodynamics, organ function, and survival in an ovine septic shock model. Design: Randomized animal study. Setting: University hospital animal research laboratory. Subjects: Forty-six adult female sheep. Interventions: Fecal peritonitis was induced in the anesthetized, mechanically ventilated, fluid-resuscitated sheep, and they were randomized in two successive phases. Three late-intervention groups (each n = 6) received IV selepressin (1 pmol/kg/min), arginine vasopressin (0.25 pmol [0.1 mU]/kg/min), or norepinephrine (3 nmol [0.5 μg]/kg/min) when mean arterial pressure remained less than 70 mm Hg despite fluid challenge; study drugs were thereafter titrated to keep mean arterial pressure at 70–80 mm Hg. Three early-intervention groups (each n = 7) received selepressin, arginine vasopressin, or norepinephrine at the same initial infusion rates as for the late intervention, but already when mean arterial pressure had decreased by 10% from baseline; doses were then titrated as for the late intervention. A control group (n = 7) received saline. All animals were observed until death or for a maximum of 30 hours. Measurements and Main Results: In addition to hemodynamic and organ function assessment, plasma interleukin-6 and nitrite/nitrate levels were measured. In the late-intervention groups, selepressin delayed the decrease in mean arterial pressure and was associated with lower lung wet/dry weight ratios than in the other two groups. In the early-intervention groups, selepressin maintained mean arterial pressure and cardiac index better than arginine vasopressin or norepinephrine, slowed the increase in blood lactate levels, and was associated with less lung edema, lower cumulative fluid balance, and lower interleukin-6 and nitrite/nitrate levels. Selepressin-treated animals survived longer than the other animals. Conclusions: In this clinically relevant model, selepressin, a selective V1A receptor agonist, was superior to arginine vasopressin and to norepinephrine in the treatment of septic shock, especially when administered early.

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Excellence in Critical Care Units

No abstract available

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Open Lung Approach for the Acute Respiratory Distress Syndrome: A Pilot, Randomized Controlled Trial*

Objective: The open lung approach is a mechanical ventilation strategy involving lung recruitment and a decremental positive end-expiratory pressure trial. We compared the Acute Respiratory Distress Syndrome network protocol using low levels of positive end-expiratory pressure with open lung approach resulting in moderate to high levels of positive end-expiratory pressure for the management of established moderate/severe acute respiratory distress syndrome. Design: A prospective, multicenter, pilot, randomized controlled trial. Setting: A network of 20 multidisciplinary ICUs. Patients: Patients meeting the American-European Consensus Conference definition for acute respiratory distress syndrome were considered for the study. Interventions: At 12-36 hours after acute respiratory distress syndrome onset, patients were assessed under standardized ventilator settings (FIO2≥0.5, positive end-expiratory pressure ≥10 cm H2O). If Pao2/FIO2 ratio remained less than or equal to 200 mm Hg, patients were randomized to open lung approach or Acute Respiratory Distress Syndrome network protocol. All patients were ventilated with a tidal volume of 4 to 8 ml/kg predicted body weight. Measurements and Main Results: From 1,874 screened patients with acute respiratory distress syndrome, 200 were randomized: 99 to open lung approach and 101 to Acute Respiratory Distress Syndrome network protocol. Main outcome measures were 60-day and ICU mortalities, and ventilator-free days. Mortality at day-60 (29% open lung approach vs. 33% Acute Respiratory Distress Syndrome Network protocol, p = 0.18, log rank test), ICU mortality (25% open lung approach vs. 30% Acute Respiratory Distress Syndrome network protocol, p = 0.53 Fisher's exact test), and ventilator-free days (8 [0-20] open lung approach vs. 7 [0-20] d Acute Respiratory Distress Syndrome network protocol, p = 0.53 Wilcoxon rank test) were not significantly different. Airway driving pressure (plateau pressure - positive end-expiratory pressure) and PaO2/FIO2 improved significantly at 24, 48 and 72 hours in patients in open lung approach compared with patients in Acute Respiratory Distress Syndrome network protocol. Barotrauma rate was similar in both groups. Conclusions: In patients with established acute respiratory distress syndrome, open lung approach improved oxygenation and driving pressure, without detrimental effects on mortality, ventilator-free days, or barotrauma. This pilot study supports the need for a large, multicenter trial using recruitment maneuvers and a decremental positive end-expiratory pressure trial in persistent acute respiratory distress syndrome.

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Shining a Light on the Problem of Circadian Phase Disruption in the Critically Ill*

No abstract available

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Neutrophil Toll-Like Receptor 9 Expression and the Systemic Inflammatory Response in Acetaminophen-Induced Acute Liver Failure

Objectives: There is a marked propensity for patients with acetaminophen-induced acute liver failure to develop sepsis, which may culminate in multiple organ failure and death. Toll-like receptors sense pathogens and induce inflammatory responses, but whether this is protective or detrimental in acetaminophen-induced acute liver failure remains unknown. Design, Setting, and Patients: We assessed Toll-like receptor expression on circulating neutrophils and their function in 24 patients with acetaminophen-induced acute liver failure and compared with 10 healthy controls. Interventions: Neutrophil Toll-like receptor 2, -4, and -9 expression and cytokine production and function were studied ex vivo at baseline and following stimulation with lipopolysaccharide, oligodeoxynucleotides, ammonium chloride, and interleukin-8. To examine the influence of acetaminophen-induced acute liver failure plasma and endogenous DNA on Toll-like receptors-9 expression, healthy neutrophils were incubated with acetaminophen-induced acute liver failure plasma with and without deoxyribonuclease-I. Measurements and Main Results: Circulating neutrophil Toll-like receptor 9 expression was increased in acetaminophen-induced acute liver failure on day 1 compared with healthy controls (p = 0.0002), whereas Toll-like receptor 4 expression was decreased compared with healthy controls (p

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Delayed Rapid Response Team Activation Is Associated With Increased Hospital Mortality, Morbidity, and Length of Stay in a Tertiary Care Institution*

Objective: To identify whether delays in rapid response team activation contributed to worse patient outcomes (mortality and morbidity). Design: Retrospective observational cohort study including all rapid response team activations in 2012. Setting: Tertiary academic medical center. Patients: All those 18 years old or older who had a rapid response team call activated. Vital sign data were abstracted from individual patient electronic medical records for the 24 hours before the rapid response team activation took place. Patients were considered to have a delayed rapid response team activation if more than 1 hour passed between the first appearance in the record of an abnormal vital sign meeting rapid response team criteria and the activation of an rapid response team. Interventions: None. Measurements and Main Results: A total of 1,725 patients were included in the analysis. Data were compared between those who had a delayed rapid response team activation and those who did not. Fifty seven percent patients met the definition of delayed rapid response team activation. Patients in high-frequency physiologic monitored environments were more likely to experience delay than their floor counterparts. In the no-delay group, the most common reasons for rapid response team activation were tachycardia/bradycardia at 29% (217/748), respiratory distress/low SpO2 at 28% (213/748), and altered level of consciousness at 23% (170/748) compared with respiratory distress/low SpO2 at 43% (423/977), tachycardia/bradycardia at 33% (327/977), and hypotension at 27% (261/977) in the delayed group. The group with no delay had a higher proportion of rapid response team calls between 8:00 and 16:00, whereas those with delay had a higher proportion of calls between midnight and 08:00. The delayed group had higher hospital mortality (15% vs 8%; adjusted odds ratio, 1.6; p = 0.005); 30-day mortality (20% vs 13%; adjusted odds ratio, 1.4; p = 0.02); and hospital length of stay (7 vs 6 d; relative prolongation, 1.10; p = 0.02) compared with the no-delay group. Conclusions: Delays in rapid response team activation occur frequently and are independently associated with worse patient mortality and morbidity outcomes.

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No abstract available

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Population Pharmacokinetics of Fentanyl in the Critically Ill*

Objective: To characterize fentanyl population pharmacokinetics in patients with critical illness and identify patient characteristics associated with altered fentanyl concentrations. Design: Prospective cohort study. Setting: Medical and surgical ICUs in a large tertiary care hospital in the United States. Patients: Patients with acute respiratory failure and/or shock who received fentanyl during the first 5 days of their ICU stay. Measurements and Main Results: We collected clinical and hourly drug administration data and measured fentanyl concentrations in plasma collected once daily for up to 5 days after enrollment. Among 337 patients, the mean duration of infusion was 58 hours at a median rate of 100 μg/hr. Using a nonlinear mixed-effects model implemented by NONMEM, we found that fentanyl pharmacokinetics were best described by a two-compartment model in which weight, severe liver disease, and congestive heart failure most affected fentanyl concentrations. For a patient population with a mean weight of 92 kg and no history of severe liver disease or congestive heart failure, the final model, which performed well in repeated 10-fold cross-validation, estimated total clearance, intercompartmental clearance (Q), and volumes of distribution for the central (V1) and peripheral compartments (V2) to be 35 L/hr (95% CI, 32–39 L/hr), 55 L/hr (95% CI, 42–68 L/hr), 203 L (95% CI, 140–266 L), and 523 L (95% CI, 428–618 L), respectively. Severity of illness was marginally associated with fentanyl pharmacokinetics but did not improve the model fit after liver and heart diseases were included. Conclusions: In this study, fentanyl pharmacokinetics during critical illness were strongly influenced by severe liver disease, congestive heart failure, and weight, factors that should be considered when dosing fentanyl in the ICU. Future studies are needed to determine if data-driven fentanyl dosing algorithms can improve outcomes for ICU patients.

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Should We Embrace the “Open Lung” Approach?*

No abstract available

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The Association Between Low Admission Peak Plasma Creatinine Concentration and In-Hospital Mortality in Patients Admitted to Intensive Care in Australia and New Zealand*

Objective: To evaluate the independent association between low peak admission plasma creatinine concentrations and in-hospital mortality in patients requiring critical care in Australia and New Zealand. Design: Multicenter, binational, retrospective cohort study. Setting: Data were extracted from the Australian and New Zealand Intensive Care Society Centre for Outcome and Resource Evaluation adult patient database. Patients: All available records for the period 2000 to 2013 were utilized. The following exclusion criteria were applied: all readmission episodes (within the same hospital stay), missing in-hospital mortality, admission post kidney transplantation, chronic renal replacement therapy (hemodialysis or peritoneal dialysis), and missing peak plasma creatinine concentration. Demographic, anthropometric, admission, illness severity, laboratory, and outcome data were then extracted. Patients were categorized on the basis of their peak (maximum) plasma creatinine concentration recorded in the first 24 hours of ICU admission. Illness severity–adjusted associations with in-hospital mortality relative to a reference category of 70–79 μmol/L were then determined using multivariate logistic regression. Interventions: Nil. Measurements and Main Results: Data pertaining to 1,250,449 admissions were available for the study period. Following exclusions, 1,045,718 patients were included. Regression analysis identified that peak plasma creatinine concentrations less than 60 μmol/L measured in the first 24 hours after ICU admission imply a steadily increasing adjusted in-hospital mortality risk. In cases where this value is markedly low (

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Lung Protective Ventilator Strategies: Beyond Scaling Tidal Volumes to Ideal Lung Size*

No abstract available

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Organ System Network Disruption in Nonsurvivors of Critically Ill Patients

Objectives: As interactions of each organ system have been conceptually known to play an important role during life-threatening conditions, we quantitatively evaluated the organ system interactions in critically ill patients and examined the difference in the organ system network structure between the survivors and the nonsurvivors. Design: Prospective observational study. Settings: An ICU of a university hospital. Patients: Two hundred and eighty-two patients who were admitted to the ICU. Interventions: Blood samples were obtained at ICU admission. Measurements and Main Results: We analyzed the associations among nine representative laboratory variables of each organ system using network analysis. We compared the network structure of the variables in the 40 nonsurvivors with that in the 40 survivors. Their baseline characteristics, including the degree of organ dysfunction, were matched using propensity score matching method. Network structure was quantitatively evaluated using edge (significant correlation among variables evaluated by the p value), weight (connective strength of edge evaluated by coefficient), and cluster (group with tight connection evaluated by edge betweenness). The number of edges among the nine variables was significantly fewer for the nonsurvivors than for the severity-matched survivors (3 vs 12; p = 0.035). The mean weight of edges was significantly smaller for the nonsurvivors (0.055 vs 0.119; p = 0.007). The nine laboratory variables for the nonsurvivors were divided into a significantly larger number of clusters (7 vs 2; p = 0.001). Statistical conclusions were preserved with Bonferroni multiple comparison procedure. These findings were consistently observed in comparison of the 40 nonsurvivors with all the survivors. Conclusions: This study, as a preliminary proof-of-concept, quantitatively demonstrated a more disrupted network structure of organ systems in the nonsurvivors compared with that in the survivors. These observations suggest the necessity of assessment for organ system interactions to evaluate critically ill patients.

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Online Editorial Board Acknowledgment

No abstract available

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Volume Delivered During Recruitment Maneuver Predicts Lung Stress in Acute Respiratory Distress Syndrome*

Objective: Global lung stress varies considerably with low tidal volume ventilation for acute respiratory distress syndrome. High stress despite low tidal volumes may worsen lung injury and increase risk of death. No widely available parameter exists to assess global lung stress. We aimed to determine whether the volume delivered during a recruitment maneuver (VRM) is inversely associated with lung stress and mortality in acute respiratory distress syndrome. Design: Substudy of an acute respiratory distress syndrome clinical trial on esophageal pressure-guided positive end-expiratory pressure titration. Setting: U.S. academic medical center. Patients: Forty-two patients with acute respiratory distress syndrome in whom airflow, airway pressure, and esophageal pressure were recorded during the recruitment maneuver. Interventions: A single recruitment maneuver was performed before initiating protocol-directed ventilator management. Recruitment maneuvers consisted of a 30-second breath hold at 40 cm H2O airway pressure under heavy sedation or paralysis. VRM was calculated by integrating the flow-time waveform during the maneuver. End-inspiratory stress was defined as the transpulmonary (airway minus esophageal) pressure during end-inspiratory pause of a tidal breath and tidal stress as the transpulmonary pressure difference between end-inspiratory and end-expiratory pauses. Measurements and Main Results: VRM ranged between 7.4 and 34.7 mL/kg predicted body weight. Lower VRM predicted high end-inspiratory and tidal lung stress (end-inspiratory: β = –0.449; 95% CI, –0.664 to –0.234; p

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Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination [ARTICLE]

Although RNA polymerase II (Pol II) productively transcribes very long genes in vivo, transcription through extragenic sequences often terminates in the promoter-proximal region and the nascent RNA is degraded. Mechanisms that induce early termination and RNA degradation are not well understood in multicellular organisms. Here, we present evidence that the suppressor of sable [su(s)] regulatory pathway of Drosophila melanogaster plays a role in this process. We previously showed that Su(s) promotes exosome-mediated degradation of transcripts from endogenous repeated elements at an Hsp70 locus (Hsp70-αβ elements). In this report, we identify Wdr82 as a component of this process and show that it works with Su(s) to inhibit Pol II elongation through Hsp70-αβ elements. Furthermore, we show that the unstable transcripts produced during this process are polyadenylated at heterogeneous sites that lack canonical polyadenylation signals. We define two distinct regions that mediate this regulation. These results indicate that the Su(s) pathway promotes RNA degradation and transcription termination through a novel mechanism.

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Watson-Crick-like pairs in CCUG repeats: evidence for tautomeric shifts or protonation [ARTICLE]

RNA transcripts that include expanded CCUG repeats are associated with myotonic dystrophy type 2. Crystal structures of two CCUG-containing oligomers show that the RNA strands associate into slipped duplexes that contain noncanonical C–U pairs that have apparently undergone tautomeric transition or protonation resulting in an unusual Watson–Crick-like pairing. The overhanging ends of the duplexes interact forming U–U pairs, which also show tautomerism. Duplexes consisting of CCUG repeats are thermodynamically less stable than the trinucleotide repeats involved in the TRED genetic disorders, but introducing LNA residues increases their stability and raises the melting temperature of the studied oligomers by ~10°C, allowing detailed crystallographic studies. Quantum mechanical calculations were performed to test the possibility of the tautomeric transitions or protonation within the noncanonical pairs. The results indicate that tautomeric or ionic shifts of nucleobases can manifest themselves in biological systems, supplementing the canonical "rules of engagement."

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High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases [ARTICLE]

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling.

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An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions [ARTICLE]

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (–H) and –amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.

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Corrigendum: Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins [CORRIGENDUM]

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Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism [ARTICLE]

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2^m), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k_cat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the k_cat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

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Long-range RNA pairings contribute to mutually exclusive splicing [ARTICLE]

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.

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Cytoplasmic poly(A) binding protein-1 binds to genomically encoded sequences within mammalian mRNAs [ARTICLE]

The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3' poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5' UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5' UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions.

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Applied Sciences, Vol. 5, Pages 1803-1836: Sequentially Palladium-Catalyzed Processes in One-Pot Syntheses of Heterocycles

Sequentially Pd-catalyzed processes are excellent entries to heterocycle synthesis. The broad mechanistic variety combined with often very mild reaction conditions allow the concatenation of elementary organic and organometallic steps to novel sequences in the sense of one-pot domino and multicomponent reactions. Given the numerous opportunities of alkyne coordination and their Pd-mediated transformations, alkynylation and carbometallation play a key role, both for purely organometallic sequences as well as in those processes that are intercepted by cyclocondensation. Pd-catalyzed aminations also find more and more entry into novel heterocycle syntheses based upon this theme.

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Applied Sciences, Vol. 5, Pages 1869-1881: Kinetic Adsorption Study of Silver Nanoparticles on Natural Zeolite: Experimental and Theoretical Models

In this research, the adsorption capacity of Ag nanoparticles on natural zeolite from Oaxaca is presented. In order to describe the adsorption mechanism of silver nanoparticles on zeolite, experimental adsorption models for Ag ions and Ag nanoparticles were carried out. These experimental data obtained by the atomic absorption spectrophotometry technique were compared with theoretical models such as Lagergren first-order, pseudo-second-order, Elovich, and intraparticle diffusion. Correlation factors R2 of the order of 0.99 were observed. Analysis by transmission electron microscopy describes the distribution of the silver nanoparticles on the zeolite outer surface. Additionally, a chemical characterization of the material was carried out through a dilution process with lithium metaborate. An average value of 9.3 in the Si/Al ratio was observed. Factors such as the adsorption behavior of the silver ions and the Si/Al ratio of the zeolite are very important to support the theoretical models and establish the adsorption mechanism of Ag nanoparticles on natural zeolite.

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Marine Drugs, Vol. 13, Pages 7419-7432: Structure-Activity Relationship and in Vivo Anti-Tumor Evaluations of Dictyoceratin-A and -C, Hypoxia-Selective Growth Inhibitors from Marine Sponge

Oral dictyoceratin-C (1) and A (2), hypoxia-selective growth inhibitors, showed potent in vivo antitumor effects in mice subcutaneously inoculated with sarcoma S180 cells. Structurally modified analogs were synthesized to assess the structure–activity relationship of the natural compounds 1 and 2 isolated from a marine sponge. Biological evaluation of these analogs showed that the exo-olefin and hydroxyl and methyl ester moieties were important for the hypoxia-selective growth inhibitory activities of 1 and 2. Thus far, only substitution of the methyl ester with propargyl amide in 1 was found to be effective for the synthesis of probe molecules for target identification.

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Applied Sciences, Vol. 5, Pages 1846-1868: Numerical Simulation of Laminar Forced Convection of Pin-Fin Heat-Sink Array in a Channel by Using Porous Approach

This work used a porous approach model to numerically investigate the fluid flow and heat transfer characteristics of the pin-fin heat-sink array in a rectangular channel with in-line arrangement. The air flow through the channel was laminar. The pin-fin heat sinks with various porosities and pin-fin numbers were employed. The relative center-to-center longitudinal and transverse distances between adjacent heat sinks were changed. The results indicate that the Nusselt number of various heat-sink arrays increased with decreasing the relative center-to-center transverse distance, but not varied with the relative center-to-center longitudinal distance. For the typical pin-fin heat-sink arrays, the Nusselt number changed slightly for the heat sinks with 0.358–0.556 porosity, but increased by 11.7%–24.8% when the porosity increased from 0.556 to 0.750, and then dropped obviously when the porosity exceeded 0.750. Increasing the number of pin fins continuously could increase Nusselt number. However, when the number of pin fins was large, the Nusselt number increased with the number of pin fins slowly. The present numerical simulation has been validated by the typical experiment. Finally, a semi-empirical correlation of Nusselt number for each heat sink in the heat-sink array was proposed.

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Molecules, Vol. 20, Pages 22534-22545: A Nanostructured Lipid System as a Strategy to Improve the in Vitro Antibacterial Activity of Copper(II) Complexes

The aim of this study was to construct a nanostructured lipid system as a strategy to improve the in vitro antibacterial activity of copper(II) complexes. New compounds with the general formulae [CuX2(INH)2]·nH2O (X = Cl− and n = 1 (1); X = NCS− and n = 5 (2); X = NCO− and n = 4 (3); INH = isoniazid, a drug widely used to treat tuberculosis) derived from the reaction between the copper(II) chloride and isoniazid in the presence or absence of pseudohalide ions (NCS− or NCO−) were synthesized and characterized by infrared spectrometry, electronic absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, elemental analysis, melting points and complexometry with 2,2′,2′′,2′′′-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA). The characterization techniques allowed us to confirm the formation of the copper(II) complexes. The Cu(II) complexes were loaded into microemulsion (MEs) composed of 10% phase oil (cholesterol), 10% surfactant [soy oleate and Brij® 58 (1:2)] and 80% aqueous phase (phosphate buffer pH = 7.4) prepared by sonication. The Cu(II) complex-loaded MEs displayed sizes ranging from 158.0 ± 1.060 to 212.6 ± 1.539 nm, whereas the polydispersity index (PDI) ranged from 0.218 ± 0.007 to 0.284 ± 0.034. The antibacterial activity of the free compounds and those that were loaded into the MEs against Staphylococcus aureus ATCC® 25923 and Escherichia coli ATCC® 25922, as evaluated by a microdilution technique, and the cytotoxicity index (IC50) against the Vero cell line (ATCC® CCL-81TM) were used to calculate the selectivity index (SI). Among the free compounds, only compound 2 (MIC 500 μg/mL) showed activity for S. aureus. After loading the compounds into the MEs, the antibacterial activity of compounds 1, 2 and 3 was significantly increased against E. coli (MIC's 125, 125 and 500 μg/mL, respectively) and S. aureus (MICs 250, 500 and 125 μg/mL, respectively). The loaded compounds were less toxic against the Vero cell line, especially compound 1 (IC50 from 109.5 to 319.3 μg/mL). The compound 2- and 3-loaded MEs displayed the best SI for E. coli and S. aureus, respectively. These results indicated that the Cu(II) complex-loaded MEs were considerably more selective than the free compounds, in some cases, up to 40 times higher.

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Energies, Vol. 8, Pages 14182-14196: Waste-to-Energy in China: Key Challenges and Opportunities

China—the largest developing country in the world—is experiencing both rapid economic maturation and large-scale urbanization. These situations have led to waste disposal problems, and the need to identify alternative energy sources. Waste-to-energy (WTE) conversion processes, a source of renewable energy, are expected to play an increasingly important role in China's sustainable management of municipal solid waste (MSW). The purpose of this research is to investigate the key problems and opportunities associated with WTE, to provide recommendations for the government. This paper begins by describing China's current MSW management situation and analyzing its waste disposal problems. The major challenges associated with China's WTE incineration are then discussed from economic, environmental and social points of view. These include the high costs associated with constructing necessary facilities, the susceptibility of facilities to corrosion, the lower heating value of China's MSW, air pollutant emissions and especially public opposition to WTE incineration. Since discarded waste can be used to produce energy for electricity and heat—thus reducing its volume and the production of greenhouse gas (GHG) emissions—with government policies and financial incentives, the use of WTE incineration as a renewable energy source and part of a sustainable waste management strategy will be of increasing importance in the future. The paper concludes by summarizing the management, economic and social benefits that could be derived from developing the country's domestic capacity for producing the needed incineration equipment, improving source separation capabilities, standardizing regulatory and legal responsibilities and undertaking more effective public consultation processes.

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Applied Sciences, Vol. 5, Pages 1837-1845: High Power Diode-Side-Pumped Q-Switched Nd:YAG Solid-State Laser with a Thermoelectric Cooler

A diode-side-pumped, high-energy, high-beam-quality, pulsed solid-state Nd3+:Y3Al5O12 (Nd:YAG) laser with a thermoelectric cooler (TEC) is investigated in this study. The pump laser was a pulsed laser diode array with maximum peak power of 15 kW. A 350 mJ laser pulse was obtained with a wavelength of 1064 nm, a pulse duration of 10 ns, a total electrical-to-optical efficiency of 7.5%, a relative stability of output energy of 5%, and a beam quality of M2 < 4.

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Energies, Vol. 8, Pages 14197-14218: Designing an Incentive Contract Menu for Sustaining the Electricity Market

This paper designs an incentive contract menu to achieve long-term stability for electricity prices in a day-ahead electricity market. A bi-level Stackelberg game model is proposed to search for the optimal incentive mechanism under a one-leader and multi-followers gaming framework. A multi-agent simulation platform was developed to investigate the effectiveness of the incentive mechanism using an independent system operator (ISO) and multiple power generating companies (GenCos). Further, a Q-learning approach was implemented to analyze and assess the response of GenCos to the incentive menu. Numerical examples are provided to demonstrate the effectiveness of the incentive contract.

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Catalysts, Vol. 5, Pages 2161-2222: Zeolite Membranes in Catalysis—From Separate Units to Particle Coatings

Literature on zeolite membranes in catalytic reactions is reviewed and categorized according to membrane location. From this perspective, the classification is as follows: (i) membranes spatially decoupled from the reaction zone; (ii) packed bed membrane reactors; (iii) catalytic membrane reactors and (iv) zeolite capsuled catalyst particles. Each of the resulting four chapters is subdivided by the kind of reactions performed. Over the whole sum of references, the advantage of zeolite membranes in catalytic reactions in terms of conversion, selectivity or yield is evident. Furthermore, zeolite membrane preparation, separation principles as well as basic considerations on membrane reactors are discussed.

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IJMS, Vol. 16, Pages 29948-29970: Transcriptome Characterization for Non-Model Endangered Lycaenids, Protantigius superans and Spindasis takanosis, Using Illumina HiSeq 2500 Sequencing

The Lycaenidae butterflies, Protantigius superans and Spindasis takanosis, are endangered insects in Korea known for their symbiotic association with ants. However, necessary genomic and transcriptomics data are lacking in these species, limiting conservation efforts. In this study, the P. superans and S. takanosis transcriptomes were deciphered using Illumina HiSeq 2500 sequencing. The P. superans and S. takanosis transcriptome data included a total of 254,340,693 and 245,110,582 clean reads assembled into 159,074 and 170,449 contigs and 107,950 and 121,140 unigenes, respectively. BLASTX hits (E-value of 1.0 × 10−5) against the known protein databases annotated a total of 46,754 and 51,908 transcripts for P. superans and S. takanosis. Approximately 41.25% and 38.68% of the unigenes for P. superans and S. takanosis found homologous sequences in Protostome DB (PANM-DB). BLAST2GO analysis confirmed 18,611 unigenes representing Gene Ontology (GO) terms and a total of 5259 unigenes assigned to 116 pathways for P. superans. For S. takanosis, a total of 6697 unigenes were assigned to 119 pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Additionally, 382,164 and 390,516 Simple Sequence Repeats (SSRs) were compiled from the unigenes of P. superans and S. takanosis, respectively. This is the first report to record new genes and their utilization for conservation of lycaenid species population and as a reference information for closely related species.

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IJMS, Vol. 16, Pages 29980-29995: Dietary Compound Kaempferol Inhibits Airway Thickening Induced by Allergic Reaction in a Bovine Serum Albumin-Induced Model of Asthma

Asthma is characterized by aberrant airways including epithelial thickening, goblet cell hyperplasia, and smooth muscle hypertrophy within the airway wall. The current study examined whether kaempferol inhibited mast cell degranulation and prostaglandin (PG) release leading to the development of aberrant airways, using an in vitro model of dinitrophenylated bovine serum albumin (DNP-BSA)-sensitized rat basophilic leukemia (RBL-2H3) mast cells and an in vivo model of BSA-challenged asthmatic mice. Nontoxic kaempferol at 10–20 μM suppressed β-hexosaminidase release and cyclooxygenase 2 (COX2)-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitized mast cells. Oral administration of ≤20 mg/kg kaempferol blocked bovine serum albumin (BSA) inhalation-induced epithelial cell excrescence and smooth muscle hypertrophy by attenuating the induction of COX2 and the formation of PGD2 and PGF2α, together with reducing the anti-α-smooth muscle actin (α-SMA) expression in mouse airways. Kaempferol deterred the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). Furthermore, the antigen-challenged activation of Syk-phospholipase Cγ (PLCγ) pathway was dampened in kaempferol-supplemented mast cells. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signaling and PKCμ-ERK-cPLA2-COX2 signaling in antigen-exposed mast cells. Thus, kaempferol may be a potent anti-allergic compound targeting allergic asthma typical of airway hyperplasia and hypertrophy.

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