Oxidative stress via hydrogen peroxide and menadione does not induce the secretion of IGFBP-5 in primary rat hepatocytes

Conference abstract describing how oxidative stress via hydrogen peroxide and menadione does not induce the secretion of IGFBP-5 in primary rat hepatocytes. Presented at the 2010 annual congress of the british toxicology societ

Insulin-like growth factor binding protein-5 as a biomarker for detection of early liver disease

Study identifying an Insulin-like growth factor binding protein-5 as a biomarker for detection of early liver disease presented at the annual congress of the british toxicology societ

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused 3D human liver bioreactor

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t1/2), rate of elimination (kel), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h-1, 6.6x10-5 L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver. Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t[subscript 1/2]), rate of elimination (k[subscript el]), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h[superscript -1], 6.6x10[superscript -5] L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-73 Microphysiological Systems W911NF-12-2-0039)National Institutes of Health (U.S.) (5-UH2-TR000496)Massachusetts Institute of Technology. Center for Environmental Health Sciences (P30-ES002109

Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K+ channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3

Pranlukast is a novel small molecule activator of the two-pore domain potassium channel TREK2

TREK2 (KCNK10, K2P10.1) is a two-pore domain potassium (K2P) channel and a potential target for the treatment of pain. Like the majority of the K2P superfamily, there is currently a lack of useful pharmacological tools to study TREK2. Here we present a strategy for identifying novel TREK2 activators. A cell-based thallium flux assay was developed and used to screen a library of drug-like molecules, from which we identified the CysLT1 antagonist Pranlukast as a novel activator of TREK2. This compound was selective for TREK2 versus TREK1 and showed no activity at TRAAK. Pranlukast was also screened against other members of the K2P superfamily. Several close analogues of Pranlukast and other CysLT1 antagonists were also tested for their ability to activate K2P channels. Consistent with previous work, structure activity relationships showed that subtle structural changes to these analogues completely attenuated the activation of TREK2, whereas for TREK1, analogues moved from activators to inhibitors. Pranlukast's activity was also confirmed using whole-cell patch clamp electrophysiology. Studies using mutant forms of TREK2 suggest Pranlukast does not bind in the K2P modulator pocket or the BL-1249 binding site. Pranlukast therefore represents a novel tool by which to study the mechanism of TREK2 activation

Metabolic and evolutionary insights into the closely-related species Streptomyces coelicolor and Streptomyces lividans deduced from high-resolution comparative genomic hybridization

Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Integrated gut/liver microphysiological systems elucidates inflammatory inter-tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk.National Institutes of Health (U.S.) (grant UH3TR00069)United States. Defense Advanced Research Projects Agency (grant Microphysiological Systems Program (W911NF-12-2-00)