Towards an Electrochemical Immunosensor System with Temperature Control for Cytokine Detection.

The cytokine interleukin-13 (IL-13) plays a major role in airway inflammation and is a target of new anti-asthmatic drugs. Hence, IL-13 determination could be interesting in assessing therapy success. Thus, in this work an electrochemical immunosensor for IL-13 was developed and integrated into a fluidic system with temperature control for read-out. Therefore, two sets of results are presented. First, the sensor was set up in sandwich format on single-walled carbon nanotube electrodes and was read out by applying the hydrogen peroxide⁻hydroquinone⁻horseradish peroxidase (HRP) system. Second, a fluidic system was built up with an integrated heating function realized by Peltier elements that allowed a temperature-controlled read-out of the immunosensor in order to study the influence of temperature on the amperometric read-out. The sensor was characterized at the temperature optimum of HRP at 30 °C and at 12 °C as a reference for lower performance. These results were compared to a measurement without temperature control. At the optimum operation temperature of 30 °C, the highest sensitivity (slope) was obtained compared to lower temperatures and a limit of detection of 5.4 ng/mL of IL-13 was calculated. Taken together, this approach is a first step towards an automated electrochemical immunosensor platform and shows the potential of a temperature-controlled read-out.

Probing the evolution, ecology and physiology of marine protists using transcriptomics.

Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

Simulation for Maintenance of Certification.

Maintenance of certification (MOC) is a process through which practitioners are able to show continuing competence in their areas of expertise. Simulation plays an increasingly important role in the assessment of students and residents, as well as in the initial practice certification for health care professionals. The use of simulation as an assessment tool in MOC has been sluggish to be universally accepted. This article discusses the role of simulation in health care education, how simulation might be effectively applied in the MOC process, and the future role of simulation in the MOC process.

Dexmedetomidine Does Not Affect Evoked Potentials During Spine Surgery.

BACKGROUND: The effect of dexmedetomidine on evoked potentials (EPs) has not been elucidated. We aimed to investigate the effect of dexmedetomidine on somatosensory, motor, and visual EPs. METHODS: After IRB approval, 40 adult patients scheduled for elective spine surgery using total IV anesthesia with propofol and remifentanil were randomly assigned to receive either dexmedetomidine (n = 20) or placebo (n = 20) in a double-blind, placebo-controlled trial. After obtaining informed consent, positioning, and baseline EPs recording, patients were randomly assigned to either IV dexmedetomidine 0.6 µg/kg infused over 10 minutes, followed by 0.6 µg/kg/h, or a corresponding volume of IV normal saline (placebo). EP measures at 60 ± 30 minutes after initiation of study drug were defined as T1 and at 150 ± 30 minutes were defined as T2. Changes from baseline to T1 (primary end point) and from baseline to T2 (secondary end point) in EP latencies (milliseconds) and amplitudes (microvolts) were compared between groups. Data presented as mean ± SD (95% confidence interval). RESULTS: Data from 40 patients (dexmedetomidine: n = 20; age, 54 ± 3 years; 10 males; placebo: n = 20; age, 52 ± 2 years; 5 males) were analyzed. There was no difference between dexmedetomidine versus placebo groups in primary end points: change of somatosensory EPs at T1, latency: 0.01 ± 1.3 (-0.64, 0.65) vs 0.01 ± 1.3 (-0.64, 0.65), P = 0.43 (-1.24, 0.45); amplitude: 0.03 ± 0.14 (-0.06, 0.02) vs -0.01 ± 0.13 (-0.07, 0.05), P = 0.76 (-0.074, 0.1); motor EPs amplitude at T1: 65.1 ± 194.8 (-35, 165; n = 18) vs 109.2 ± 241.4 (-24, 243; n = 16), P = 0.57 (-113.5, 241.57); visual EPs at T1 (right eye), amplitude: 2.3 ± 3.6 (-0.4, 5.1; n = 11) vs 0.3 ± 6.0 (-3.3, 3.9; n = 16), P = 0.38 (-6.7, 2.6); latency N1: 2.3 ± 3.6 (-0.4, 5.1) vs 0.3 ± 6.0 (-3.3, 3.9), P = 0.38 (-6.7, 2.6); latency P1: -1.6 ± 13.4 (-11.9, 8.7) vs -1.4 ± 8.1 (-6.3, 3.5), P = 0.97 (-9.3, 9.7) or secondary end points. There were no differences between right and left visual EPs either at T1 or at T2. CONCLUSIONS: In clinically relevant doses, dexmedetomidine as an adjunct to total IV anesthesia does not seem to alter EPs and therefore can be safely used during surgeries requiring monitoring of EPs.

Ambulatory surgery: is the liability risk lower?

PURPOSE OF REVIEW: To summarize the currently available data on malpractice claims related to ambulatory anesthesia and provide an insight into the emerging patterns of anesthesia liability in this practice setting. RECENT FINDINGS: At present, studies are mixed about how the continued growth of outpatient surgery will impact liability for anesthesiologists. Data derived from the ASA Closed Claims Project suggests that malpractice claims for major damaging events are less common in the outpatient settings than in inpatient settings. Correspondingly, the payment amounts for outpatient claims are significantly lower than those for inpatients. Nevertheless, nondisabling adverse events are common and involve respiratory, cardiac, equipment-related, and drug errors. In addition, the vast majority of injuries in outpatient claims was the result of substandard care and judged preventable by better monitoring. Although major incidents leading to malpractice suits are less, new liability exposure may be on the horizon, due to the changing landscape of ambulatory practice that permits care for sicker patients who require more complex surgeries. The areas of potential concern include postoperative discharge criteria, care for the obstructive sleep apnea patient, and the choice of anesthetic techniques such as neuraxial blocks and monitored anesthesia care. SUMMARY: With steady increase in outpatient surgery, anesthesiologists are confronted with new areas of liability. More data are needed to identify these risks and reduce exposure to malpractice claims.

Tailoring a stabilized variant of hydroxynitrile lyase from Arabidopsis thaliana.

The R-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface-modified variant incorporating 11 changes in the amino acids on the protein surface. Comparative characterization of the variant and the wild-type enzyme showed a broadened pH optimum towards the acidic range, along with enhancement of activity by up to twofold and significantly increased pH- and thermostabilities. The effect can most probably be explained by a shift of the isoelectic point from pH 5.1 to 4.8. Application of the variant for the synthesis of (R)-cyanohydrins in an aqueous/organic two-phase system at pH 4.5 demonstrated the high stereoselectivity and robustness of the variant relative to the wild-type enzyme, which is immediately inactivated under these conditions.

Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3.

The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.

Dimerization of human 5-lipoxygenase.

Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.

Closed claims' analysis.

The American Society of Anesthesiologists (ASA) Closed Claims database was started in 1985 to study anaesthesia injuries to improve patient safety, now containing 8954 claims with 5230 claims since 1990. Over the decades, claims for surgical anaesthesia decreased, while claims for acute and chronic pain management increased. In the 2000s, chronic pain management involved 18%, acute pain management 9% and obstetrical anaesthesia formed 8% of claims. Surgical anaesthesia claims with monitored anaesthesia care (MAC) increased in the 2000s to 10% of claims, while regional anaesthesia involved 19%. The most common complications were death (26%), nerve injury (22%) and permanent brain damage (9%). The most common damaging events due to anaesthesia in claims were regional-block-related (20%), respiratory (17%), cardiovascular (13%) and equipment-related events (10%). This review examines recent findings and clinical implications for injuries in management of the difficult airway, MAC, non-operating room locations, obstetric anaesthesia and chronic pain management.

Treatment of severe lactic acidosis during the pre-anhepatic stage of liver transplant surgery with intraoperative hemodialysis.

Severe uncompensated lactic acidosis manifesting during the pre-anhepatic stage of orthotopic liver transplant surgery is an uncommon event, but it poses serious concern because of the additional lactate production and impaired elimination by the liver that develops during the anhepatic and allograft reperfusion stages of the procedure. A man with end-stage liver disease secondary to hepatitis C and hemochromatosis and normal renal function, who developed severe lactic acidosis in the pre-anhepatic stage of liver transplantation, was treated successfully with intraoperative, continuous venovenous hemodialysis. Hemodialysis effectively corrected the patient's lactic acidosis and removed lactate, which contributed to hemodynamic stability during the anhepatic and graft reperfusion stages of his liver transplant surgery.

Life-threatening hyperkalemia during radiofrequency ablation of hepatocellular carcinoma.

A 48 year-old man with hepatitis C and cirrhosis was admitted for laparoscopic radiofrequency ablation of a large hepatocellular carcinoma. The patient's renal function tests and serum potassium level were all within normal limits. About 120 minutes into the procedure, the patient developed sudden, wide-complex tachycardia. Initial blood tests showed serum and plasma potassium level of 7 mEq/L, but no other abnormalities. The thermal destruction of large tumors during radiofrequency ablation may be associated with extensive cell breakdown and transcellular shift of potassium.

Risks of anesthesia or sedation outside the operating room: the role of the anesthesia care provider.

PURPOSE OF REVIEW: Our goal is to review the recent year's novel and relevant literature on the practice of sedation/anesthesia in the nonoperating room setting. Risk factors and outcomes were evaluated related to locations, providers, and anesthetic regimens. RECENT FINDINGS: Administration of sedation/anesthesia for patients undergoing uncomfortable or painful interventions outside the operating room is an expanding practice involving a wide variety of practitioners. With a growing emphasis on cost, efficiency, and patient satisfaction, propofol alone or in combination with other sedatives/analgesics has become popular for procedural sedation among nonanesthesiologists. Although major adverse events are rare in this setting, potentially risky complications, such as respiratory depression and desaturation, still occur and their importance cannot be neglected. In this context, the American Society of Anesthesiologists Closed Claims and the Pediatric Sedation Research Consortium databases convey some valuable data. The bulk of reported complications are related to anesthetic drug-induced respiratory depression or airway obstruction leading to hypoxemia or hypoventilation. There are several new studies highlighting the importance of capnography in detecting impending airway or respiratory adverse events. SUMMARY: The current incidence of complications associated with sedation in the nonoperating room environment remains irresolute. Although there are many studies on sedation practices in the out-of-operating room setting, high-quality studies are lacking. There are no data comparing practice outcomes between different practitioners and specialties.

R-flurbiprofen reduces neuropathic pain in rodents by restoring endogenous cannabinoids.

BACKGROUND: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPARgamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. CONCLUSION: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.

Sulindac sulfide suppresses 5-lipoxygenase at clinically relevant concentrations.

Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug's chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC(50) approximately 8-10 microM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC(50) = 18.7 microM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC(50) of 20 muM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.

5-lipoxygenase: underappreciated role of a pro-inflammatory enzyme in tumorigenesis.

Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis.

Anesthetic management of acute mesenteric ischemia in elderly patients.

Ischemic insult to the splanchnic vasculature can jeopardize bowel viability and lead to devastating consequences, including bowel necrosis and gangrene. Although acute mesenteric ischemia (AMI) may occur at any age, the elderly are most commonly affected due to their higher incidence of underlying systemic pathology, most notably atherosclerotic cardiovascular disease. Treatment options include pharmacology-based actions, endovascular, and surgical interventions. AMI remains a life-threatening condition with a mortality rate of 60% to 80%, especially if intestinal infarction has occurred and surgical intervention becomes emergent. Early recognition and an aggressive therapeutic approach are essential if the usually poor outcome is to be improved. Anesthetic management is complex and must account for comorbid disease as well as the patient's presumptive acute deterioration. Blood pressure support typically involves careful, but often massive, fluid resuscitation and may also additionally require pharmacologic support.

The risk and safety of anesthesia at remote locations: the US closed claims analysis.

PURPOSE OF REVIEW: A growing number of procedures are performed outside the operating room. In spite of their relatively noninvasive nature, serious adverse outcomes can occur. We analyzed claims from 1990 and later in the American Society of Anesthesiologists Closed Claims database to assess patterns of injury and liability associated with claims from anesthesia in remote locations (n = 87) compared with claims from operating room procedures (n = 3287). RECENT FINDINGS: Compared with operating room claims, remote location claims involved older and sicker patients (P < 0.01), with 50% of remote location claims involving monitored anesthesia care. The proportion of claims for death was increased in remote location claims [54 vs. 29% (operating room claims), P < 0.001]. Respiratory damaging events were more common in remote location claims (44 vs. 20%, P < 0.001), with inadequate oxygenation/ventilation the most common specific event (21 vs. 3% in operating room claims, P < 0.001). Remote location claims were more often judged as being preventable by better monitoring (32 vs. 8% for operating room claims, P < 0.001). CONCLUSION: Data from the American Society of Anesthesiologists, Closed Claims database suggest that anesthesia at remote locations poses a significant risk for the patient, particularly related to oversedation and inadequate oxygenation/ventilation during monitored anesthesia care. Similar anesthesia and monitoring standards and guidelines should be used in all anesthesia care areas.

Celecoxib inhibits 5-lipoxygenase.

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used in the therapy of inflammatory and painful conditions. Various COX-2-independent pharmacological effects, such as a chemo-preventive and tumor-regressive activity have been suggested, but the respective non-COX-2 targets of celecoxib are still a matter of research. We now demonstrate that celecoxib inhibits 5-lipoxygenase (5-LO), a key enzyme in leukotriene (LT) biosynthesis. Celecoxib suppressed 5-LO product formation in ionophore A23187-activated human polymorphonuclear leukocytes (IC(50) approximately 8 microM). Similarly, celecoxib inhibited LTB(4) formation in human whole blood (IC(50) approximately 27.3 microM). Direct interference of 5-LO with celecoxib was visualized by inhibition of enzyme catalysis both in cell homogenates and with purified 5-LO (IC(50) approximately 23.4 and 24.9 microM, respectively). Related lipoxygenases (12-LO and 15-LO) were not affected by celecoxib. Other COX-2 inhibitors (etoricoxib and rofecoxib) or unselective NSAIDs (non-steroidal anti-inflammatory drugs, diclofenac) failed to inhibit 5-LO. In rats which received celecoxib (i.p.), the blood LTB(4) levels were dose-dependently reduced with an ED(50) value approximately 35.2 mg/kg. Together, celecoxib is a direct inhibitor of 5-LO in vitro and in vivo. These findings provide a potential molecular basis for some of the described COX-2-independent pharmacological effects of celecoxib.