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Purpose – Globalization and the COVID (post) pandemic continue to pose significant challenges to managing employee performance across geographic locations because there is a need to simultaneously implement procedures that are standardized and yet applicable to different contexts. This study aims to describe five universal principles in performance management that can be adapted to specific contexts to address the performance management standardization vs adaptation dilemma. Design/methodology/approach – Critical literature review of evidence-based recommendations for practice. Findings – This study describe five universal principles in performance management, how they can be adapted to specific contexts around the world and actions that organizations can take to implement them: (1) cultural congruence, (2) strategic congruence, (3) performance evaluation thoroughness, (4) inclusiveness and (5) effective feedback. Originality/value – This study provide valuable and actionable knowledge for organizations facing performance management challenges around the world.

Introduction and Objectives: International studies have described an epidemiological change in patients with the hepatitis C virus (HCV), with greater involvement of young people and risk groups. The reality in Latin America, particularly in Chile, is unknown. This study aimed to epidemiologically characterize HCV patients treated in the Chilean public health system (period 2016-2021) and compare these characteristics in two periods (2016 - 2019 vs. 2020 - 2021). Materials and Methods: Historical cohort was constructed based on national data and the Hospital del Salvador registry (Santiago, Chile) (n=410). All patients diagnosed with HCV treated in the Chilean public system (2016-2019) and those treated at Hospital del Salvador (2020-2021 period) were included. It was registered: year of diagnosis, age, sex, presence of cirrhosis, HCV genotype, co-infection with hepatitis B virus (HBV) and/or HIV, need for a liver transplant, or intratreatment dead. Both periods were compared using the Mann-Whitney U test or Fisher's exact test, as appropriate. Results: 61.2% of patients were male, with a median age of 57 years. 73.5% presented genotype 1 and 11.6% genotype 4. There was a 19.3% coinfection with HIV. Only 1.4% had therapy failure at 24 weeks and 5.2% of patients underwent liver transplantation. When comparing the periods 2016 – 2019 vs. 2020 – 2021 a reduction in the median age 59 vs 49 (p

Introduction and Objectives: Acute-on-chronic liver failure (ACLF) is a severe clinical entity with organ failures and high short-term mortality. To Date, few ACLF reports have been published in Latin America. This study aimed to characterize patients with ACLF, identifying triggers, organ failure, and survival at 30, 90, and 180 days compared to patients with decompensated cirrhosis without ACLF. Materials and Methods: Retrospective study of decompensated cirrhotic patients hospitalized (between 2017-2019) in three centers in Chile. We evaluated transplant-free survival using Kaplan-Meier curves and Cox-regression. Results: 398 patients were included, a median age of 65.3±11.7-year-old, 50.5% female, 91 (22.9%) presented ACLF (8% ACLF-1, 6.3% ACLF-2, 8.6% ACLF-3); 6.6% underwent liver transplantation. ACLF patients were younger (63.6 vs. 66.0 years; p=0.045), had higher MELD-Na scores (27 [23-32] vs. 17 [13-23]; p

Introduction and Objectives: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Still, there are epidemiological and clinical data in Latin America. In Chile, this is the first study regarding HCC with a significant number of patients. This study aimed to obtain and analyze clinical and epidemiological data of Chilean patients with HCC. Materials and Methods: Multicenter study from 12 Chilean hospitals that have members of the Chilean Association of Hepatology as members of their staff. Clinical records from 2015-2021 were included. Kaplan-Meier survival curves and Cox regression analysis were obtained. Results: We obtained data from 856 patients with HCC from 12 different centers. Median age 67 years old; 58.7% male. Cirrhosis is present in 91.2% (779) of cases. Main risk factors reported: fatty liver 47.9%(410), alcohol 19.6% (68), viral 12.2%(104) and autoimmune 3.5% (30). The median MELD score was 11.7 (CI95% 11,4-12). 38% (322) were diagnosed during surveillance; this was associated with earlier BCLC stage (OR 2,6; CI95%1,9-3,4). BCLC stages at diagnosis were 0; 8,2%(69), A: 38,5%(326), B:29,9%(253), C: 15,4%(130) and D: 8,2%(69). The main initial treatments were TACE, ablation, resection, liver transplant and sorafenib in 27,4%(226), 19,3%(159), 11,4%(94), 8%(66) and 5,5%(45), respectively. 53,4%(452) pts were in Milan Criteria at diagnosis. 9,1%(78) patients got a liver transplant. Five-year survival was 24% (CI95%20-28). The main factors associated with survival are depicted in Figure 1. Conclusions: Fatty liver was remarkably the main risk factor reported for HCC in our Chilean cohort. This is a worrisome number since NAFLD is on the rise worldwide, and especially in Latin America. Surveillance is key for early detection. The liver function defined by Child-Pugh and HCC stage using BCLC staging is strongly associated with survival. Liver transplant is still a scarce treatment resource.

El trabajo aquí presentado es resultado de una análisis comparativo de dos herramientas para la creación de un cubo OLAP, el cual es un Procesamiento Analítico en Línea, que ejecuta un análisis de datos; una de las herramientas es Microsoft Analysis Services con SQL Server y la otra Microsoft Excel; para la definición del universo de datos se utilizó una base de datos experimental obtenida de una encuesta realizada por el Instituto Nacional de Estadística y Geografía (INEGI) del año 2016; con estas herramientas existentes se busca proporcionar un análisis para el mejor uso en la explotación de datos, identificando los diversos factores necesarios para la creación, desarrollo e implementación de los cubos OLAP y entre esos se encuentran identificar alguno patrones de los riesgos o errores al ser creados por parte de algunas de estas dos soluciones existentes en el mercado, los cuales se describirán en el presente trabajo.

Internet desde sus inicios y su evolución ha permitido la comunicación y colaboración de información, atravesando barreras de distancia y tiempo, convirtiéndose en el mayor recurso tecnológico usado hoy en día. La finalidad de la investigación se enfocó en la identificación de las estrategias a utilizar para implementar el Proyecto México Conectado en el estado de Tabasco, usando herramientas cualitativas y cuantitativas a las que se suma una metodología de desarrollo de procesos ágil llamada SCRUM. Algunos resultados fueron: la elaboración de dos instrumentos de recopilación de datos con setenta y ocho (78) ítems para la selección y validación de 7.272 sitios y espacios públicos propuestos para recibir la conectividad de banda ancha en la entidad. Estos sitios se agruparon en tres sectores: Gobierno, Educativo y Salud, con el propósito de construir un entorno tecnológico inclusivo para todos los ciudadanos. En las conclusiones destaca el crecimiento tecnológico que tendrá el estado, con la activación del servicio de Internet en los sitios en los próximos años y la detección de factores de exclusión de los inmuebles registrados, como la falta de energía eléctrica, seguridad física y accesos viales en condiciones difíciles.

Intensive development of infrastructure for fast processing of outsized amount of space-borne data enables now to use the satellite data for operational controlling the state of its environment. In our presentation we show some examples of analysis of processes in marine environment which are possible due to satellite data and algorithms of its processing developed in SatBaltic Project. It concerns supporting of modelling of solar energy inflow to the sea with space-borne input data, identification and analysis of sea ice cover, supporting of oil spill detection, and identification of phenomena which modify spatial distribution of the sea surface temperature.

This paper attempts to bring up the sense of vital energy in people's lives. Without a thorough planning, energy could lead a problem to wildlife as a country's energy security and also could threaten the national security of countries concerned. Choosing a source of energy for at least three factors that must be observed, namely security of supply, pricing (economical price), and the environment that are closely associated with the dimension of time and space.

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE -/- Fbn1 C1039G+/- ) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE -/- Fbn1 C1039G+/- mouse model. Female ApoE -/- Fbn1 C1039G+/ - mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 ± 1.2 vs. 8.1 ± 0.7 μm) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 ± 2% vs. 27 ± 2%) combined with a decreased end diastolic (3.1 ± 0.2 vs. 3.9 ± 0.2 mm) and end systolic diameter (1.9 ± 0.1 vs. 2.9 ± 0.2 mm). Furthermore, perivascular fibrosis (23 ± 2 vs. 30 ± 2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 ± 0.04 vs. 2.91 ± 0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The Schisandraceae family is reported to have a range of pharmacological activities, including anti-inflammatory effects. As with all herbal preparations, extracts of Schisandra species are mixtures composed of >50 lignans, especially schizandrins, deoxyschizandrins, and gomisins. In China, Schisandra sphenanthera extract (SSE) is often coadministered with immunosuppressant treatment of transplant recipients. In cases of coadministration, the potential for herb-drug interactions (HDIs) increases. Clinical studies have been used to assess HDI potential of SSE. Results demonstrated that chronic SSE administration reduced midazolam (MDZ) clearance by 52% in healthy volunteers. Although clinical studies are definitive and considered the "gold standard," these studies are impractical for routine HDI assessments. Alternatively, in vitro strategies can be used to reduce the need for clinical studies. Transporter-certified sandwich-cultured human hepatocytes (SCHHs) provide a fully integrated hepatic cell system that maintains drug clearance pathways (metabolism and transport) and key regulatory pathways constitutive active/androstane receptor and pregnane X receptor (CAR/PXR) necessary for quantitative assessment of HDI potential. Mechanistic studies conducted in SCHHs demonstrated that SSE and the more commonly used dietary supplement Schisandra chinensis extract (SCE) inhibited CYP3A4/5-mediated metabolism and induced CYP3A4 mRNA in a dose-dependent manner. SSE and SCE reduced MDZ clearance to 0.577- and 0.599-fold of solvent control, respectively, in chronically exposed SCHHs. These in vitro results agreed with SSE clinical findings and predicted a similar in vivo HDI effect with SCE exposure. These findings support the use of an SCHH system that maintains transport, metabolic, and regulatory functionality for routine HDI assessments to predict clinically relevant clearance interactions., (Copyright © 2017 by The Author(s).)

Intubating laryngeal mask airways can be used to provide continuous ventilation throughout intubation. This is a case of a morbidly obese (body mass index = 58) 65-year-old woman with T10 and T11 compression fractures. Optimal positioning for airway management was hindered by her unstable spine, minimal neck range of motion, and extreme pain with any movement. An intubating laryngeal mask airway was placed in the awake, topically anesthetized patient, and the laryngeal mask airway and endotracheal tube combination was left in place throughout surgery., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death., Methods and Results: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke., Conclusions: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to target, as it is the key negative regulator of the tumor suppressor p53, which controls the transcription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inactivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small-molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents., (©2011 AACR.)

Objective: Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques., Methods and Results: Apolipoprotein E-deficient (apoE(-/-)) mice were crossbred with animals lacking APP (APP(-/-)). After 16 weeks on a Western-type diet, apoE(-/-) and APP(-/-)/apoE(-/-) mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP(-/-)/apoE(-/-) mice as compared to apoE(-/-). Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP(-/-)/apoE(-/-) mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap., Conclusion: Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Because macrophages play an important role in the destabilization of atherosclerotic plaques, and smooth muscle cells (SMCs) contribute to plaque stability, selective clearance of macrophages in atherosclerotic plaques is a promising strategy for plaque stabilization. In the present study, we investigated the effects of fluvastatin, simvastatin, lovastatin, and pravastatin on the viability of macrophages and SMCs. All statins, except pravastatin, induced cell death of J774A.1 macrophages after 24 hours, albeit with different sensitivity. The viability of rabbit aortic SMCs was hardly affected. Fluvastatin-induced macrophage cell death was characterized as apoptosis and could be reversed by isoprenoid intermediates of the mevalonate pathway. Peritoneal macrophages from male or female mice were much more resistant to statin-induced cell death. The high sensitivity of J774A.1 macrophages to statin-induced cell death was related to the strong 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in these cells. Macrophage and SMC content of rabbit atherosclerotic plaques was not affected after in vitro treatment with fluvastatin or lovastatin for 3 days. In conclusion, fluvastatin, simvastatin, and lovastatin, but not pravastatin, can selectively induce apoptosis in J774A.1 macrophages, but not in SMCs, primary macrophages or rabbit atherosclerotic plaques. This effect was related to the degree of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the different cell types.

The HOPE and EUROPA clinical studies have shown that treatment with the angiotensin-converting enzyme (ACE) inhibitors, ramipril and perindopril, may reduce the occurrence of major cardiovascular events in patients with proven atherosclerotic disease. The recently published results of the PRoFESS and TRANSCEND trials completed the much needed information concerning the use of an angiotensin receptor blocker for patients at high risk of cardiovascular events. PROFESS compared a therapy of telmisartan 80 mg daily with placebo in patients with a recent ischemic stroke. The difference in the primary outcome of first recurrent stroke was not statistically significant between telmisartan and placebo. The secondary outcome of major cardiovascular events showed a relative risk reduction (RRR) of 7% in favour of telmisartan. This tended to be significant (p = 0.06) despite a rather short follow-up period of only 28 months. In TRANSCEND 5926 patients at high risk for cardiovascular events were randomized to a treatment with telmisartan 80 mg daily or placebo for a mean duration of follow-up of 56 months. The primary composite outcome of cardiovascular death, myocardial infarction, stroke or hospitalization for heart failure showed a non-significant 8% RRR in favour of the telmisartan treated patients. The main secondary outcome of cardiovascular death and myocardial infarction or stroke as used in the HOPE trial showed a non-significant RRR of 13% in favour of telmisartan treated patients (p = 0.068 adjusted for multiplicity of comparisons). In comparing the Kaplan-Meier curves for the endpoint of major cardiovascular events used in HOPE, EUROPA, TRANSCEND and PRoFESS, the trends are similar. Results of most of the recently published trials have been neutral.This could partly be explained by major improvements in the optimal background therapy of the patients included. Nevertheless, the results of PRoFESS and TRANSCEND do not contradict the results from previous studies with theACE inhibitors ramipril and perindopril and the ARB telmisartan.

Rupture-prone and ruptured plaques are characterized by the presence of large numbers of macrophages. N1177 is a contrast agent consisting of iodinated nanoparticles that are selectively phagocytosed by macrophages. The aim of this study was to investigate the effect of N1177 on the CT attenuation of rupture-prone and ruptured plaques in rabbits. In addition, we examined in vitro whether uptake of N1177 occurred without cytotoxic or pro-inflammatory effects on macrophages. In vitro, the viability of J774 macrophages was not affected by treatment with N1177. Moreover, N1177 had no effect on the phagocytic capacity or cytokine production of macrophages. For the in vivo experiments, 6 New Zealand White rabbits were fed a cholesterol-supplemented diet for 12-15 months, resulting in the development of large atherosclerotic plaques that resembled rupture-prone plaques in humans. In three rabbits, mechanical plaque rupture was induced by retrograde pullback of an embolic protection device. N1177 had no effect on the median density of rupture-prone plaques [35 HU (range 3-85) before injection vs. 32 HU (range 1-93) 2 h after injection of N1177; P > 0.05]. However, after induction of mechanical plaque rupture, the median density of the atherosclerotic plaques increased from 40 HU (range 6-86) before injection to 74 HU (range 14-111) 2 h after injection of N1177 (P < 0.001). Using time-of-flight static secondary ion mass spectrometry, the presence of N1177 nanoparticles was demonstrated in macrophage-rich areas of ruptured plaques, but not of non-ruptured plaques. In conclusion, our results show that N1177 is a contrast agent that can identify ruptured atherosclerotic plaques.

Aim: Transglutaminase 2 (TG2) is important for the deposition and stability of the extracellular matrix via effects on cross-linking of matrix proteins and transforming growth factor beta (TGFbeta) activity. The purpose of this study was to investigate the effect of TG2 deficiency on the composi- tion of atherosclerotic plaques., Methods: Apolipoprotein E (ApoE)(-/-) mice were crossbred with TG2(-/-) mice to obtain ApoE(-/-)TG2(-/-) mice. ApoE(-/-) and ApoE(-/-)TG2(-/-) mice were fed a Western-type diet for 16 or 30 weeks to determine the effect of TG2 deficiency on early and advanced atherosclerosis, respectively., Results: Atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice showed decreased cross-linking of matrix proteins, as well as decreased nuclear staining for phospho-Smad2/-Smad3, indicative of decreased TGFbeta activity. Compared to ApoE(-/-) mice, plaque area was decreased by 45 and 48% in ApoE(-/-)TG2(-/-) mice after 16 and 30 weeks, respectively. Sirius red staining showed a significant decrease in collagen content in early and advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice. Furthermore, there was a significant increase in macrophages in advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice., Conclusion: TG2 deficiency resulted in a decreased collagen content and increased inflammation, which are features of a more unstable plaque., (Copyright 2009 S. Karger AG, Basel.)

The ubiquitin-proteasome system is involved in the development and progression of atherosclerosis. The aim of this study was to investigate whether plaque composition is affected by proteasome function. In vitro, the potent and selective proteasome inhibitor bortezomib induced apoptosis in both cultured smooth muscle cells (SMCs) and activated macrophages. This effect was associated with increased expression of C/EBP homologous protein and cleavage of caspase-12, indicative of endoplasmic reticulum stress. The sensitivity to the proapoptotic effects of proteasome inhibition correlated with the protein synthesis rate. Proteasome inhibition in explanted atherosclerotic plaques of ApoE-deficient mice resulted in a significant decrease in SMCs and macrophages, indicating that both cell types in the atherosclerotic plaque were susceptible to the proapoptotic effects of proteasome inhibition. In vivo proteasome inhibition in ApoE-deficient mice did not affect plaque size or composition of early atherosclerotic plaques, but resulted in a significant decrease in collagen content as well as a significant enlargement of the necrotic core in advanced atherosclerotic plaques. In conclusion, our results indicate that an impaired proteasome function promotes features of a more rupture-prone plaque phenotype.

Unstable atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophage origin. Previously we and others demonstrated that macrophages disappear from atherosclerotic plaques after dietary lipid lowering. However, it remains unclear whether loss of macrophages after lipid lowering occurs via increased apoptosis, decreased macrophage replication and/or recruitment, or via a combination of both. Rabbits were fed a diet supplemented with cholesterol (0.3%) for 24 weeks followed by a normal diet for 4, 12, or 24 weeks. After 24 weeks of cholesterol supplement, plaques showed apoptosis in both macrophages and SMCs, as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Cell replication (Ki-67 immunolabeling) was predominantly present in macrophages. After 24 weeks of cholesterol withdrawal, the thickness and areas of the plaques were unchanged. Nevertheless, plaques showed a considerable loss of macrophages. This event was associated with a reduced immunoreactivity for vascular cell adhesion molecule-1 (VCAM-1) in the endothelial cells starting 4 weeks after cholesterol withdrawal. Apoptosis did not increase after lipid lowering but showed a steady decline. Apart from decreased VCAM-1 expression, a strong decrease in Ki-67 immunolabeling was observed after 12 weeks of cholesterol withdrawal. Our findings suggest that loss of macrophages in atherosclerotic plaques after dietary lipid lowering is not related to induction of macrophage apoptosis but mainly a consequence of impaired monocyte recruitment followed by decreased macrophage replication. This information is essential for understanding the effects of aggressive lipid lowering on plaque stability.

Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active-site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent K(i) for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active-site conformation nearly identical to that of non-allosteric beta-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Phagocytosis represents a mechanism used by macrophages to remove pathogens and cellular debris. Recent evidence suggests that phagocytosis is stimulated under specific conditions of stress, such as extracellular pressure and hypoxia. In the present study, we show that amino acid or glucose deprivation caused an increase in the phagocytosis of heat-inactivated Escherichia coli and Staphylococcus aureus by macrophages, but not the uptake of platelets, apoptotic cells or beads. Increased phagocytosis of bacteria could be blocked by phagocytosis inhibitors and was found to be dependent on p38 mitogen-activated protein kinase activity and scavenger receptor A. Although nutrient deprivation is a strong stimulus of autophagy, autophagosome formation was not critical for the uptake of bacteria because phagocytic clearance was not inhibited after down-regulation of the autophagy essential gene Atg7. Moreover, enhanced uptake of bacteria should not be considered as a general stress response because phagocytosis of bacteria was not stimulated after exposure of macrophages to the genotoxic agent camptothecin, heat (40 degrees C) or thapsigargin-induced endoplasmic reticulum stress. Overall, the results obtained in the present study indicate that nutrient deprivation can stimulate macrophages to fight bacterial infections.

Background and Purpose: Tachykinin NK(3) receptors are widely expressed in the mouse gastrointestinal tract but their functional role in enteric neuromuscular transmission remains unstudied in this species. We investigated the involvement of NK(3) receptors in cholinergic neurotransmission in the mouse stomach and small intestine., Experimental Approach: Muscle strips of the mouse gastric fundus and ileum were mounted in organ baths for tension recordings. Effects of NK(3) agonists and antagonists were studied on contractions to EFS of enteric nerves and to carbachol., Key Results: EFS induced frequency-dependent tetrodotoxin-sensitive contractions, which were abolished by atropine. The cholinergic contractions to EFS in the stomach were enhanced by the NK(3) antagonist SR142801, but not affected by the NK(3) agonist senktide or neurokinin B. The cholinergic contractions to EFS in the small intestine were not affected by SR142801, but dose-dependently inhibited by senktide and neurokinin B. This inhibitory effect was prevented by SR142801 but not by hexamethonium. SR142801, senktide or neurokinin B did not induce any response per se in the stomach and small intestine and did not affect contractions to carbachol., Conclusions and Implications: NK(3) receptors modulate cholinergic neurotransmission differently in the mouse stomach and small intestine. Blockade of NK(3) receptors enhanced cholinergic transmission in the stomach but not in the intestine. Activation of NK(3) receptors inhibited cholinergic transmission in the small intestine but not in the stomach. This indicates a physiological role for NK(3) receptors in mouse stomach contractility and a pathophysiological role in mouse intestinal contractility.

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis-induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single-fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 0.25-5 mg kg(-1)) or its vehicle (hydroxypropyl-beta-cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A-fibre neurons, was performed in the dorsal root ganglia L6-S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C-fibres. BCTC did not significantly affect the C-fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C-fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Adelta-fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up-regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis-induced sensitization of pelvic afferent C-fibres to CRD, while Adelta-fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Sepsis is an inflammatory condition that is associated with reduced propulsive gastrointestinal motility (ileus). A therapeutic option to treat sepsis is to promote intestinal propulsion preventing bacterial stasis, overgrowth and translocation. Recent evidence suggests that anti-oxidants improve sepsis-induced ileus. Cannabidiol, a non-psychotropic component of Cannabis sativa, exerts strong anti-oxidant and anti-inflammatory effects without binding to cannabinoid CB(1) or CB(2) receptors. Cannabidiol also regulates the activity of fatty acid amide hydrolase (FAAH) which is the main enzyme involved in endocannabinoid breakdown and which modulates gastrointestinal motility. Because of the therapeutic potential of cannabidiol in several pathologies, we investigated its effect on sepsis-induced ileus and on cannabinoid receptor and FAAH expression in the mouse intestine. Sepsis was induced by treating mice with lipopolysaccharides for 18 h. Sepsis led to a decrease in gastric emptying and intestinal transit. Cannabidiol further reduced gastrointestinal motility in septic mice but did not affect gastrointestinal motility in control mice. A low concentration of the CB(1) antagonist AM251 did not affect gastrointestinal motility in control mice but reversed the effect of cannabidiol in septic mice. Sepsis was associated with a selective upregulation of intestinal CB(1) receptors without affecting CB(2) receptor expression and with increased FAAH expression. The increase in FAAH expression was completely reversed by cannabidiol but not affected by AM251. Our results show that sepsis leads to an imbalance of the endocannabinoid system in the mouse intestine. Despite its proven anti-oxidant and anti-inflammatory properties, cannabidiol may be of limited use for the treatment of sepsis-induced ileus.

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

The recently published results of the ONTARGET trial shed a new light on the cardiovascular protection of patients at high risk of a cardiovascular event. Despite a number of trials looking at the efficacy of Angiotensin Converting Enzyme inhibitors (ACEis) or Angiotensin Receptor Blockers (ARBs) in the prevention of cardiovascular events in patients with specific high risk profiles, the question of the equivalence of ACEis and ARBs remained unanswered. The ONTARGET trial has shown that telmisartan 80 mg administered for a median duration of 4.5 years to patients at high risk of developing a major cardiovascular event, is equally effective to ramipril 10 mg. In addition, telmisartan was slightly better tolerated. The comparator ramipril has been chosen as it is currently the gold standard ACEi since the results of the HOPE study, in terms of the composite outcome of cardiovascular death, myocardial infarction and stroke. Moreover, ONTARGET is the first trial to test the hypothesis of superiority of adding an ARB (telmisartan 80 mg) to an ACEi (ramipril 10 mg) over the ACEi ramipril monotherapy in cardiovascular protection of the same broad range of high-risk patients. Surprisingly, despite a more pronounced blood pressure lowering, the combination of the two agents did not lead to an additional decrease in the number of events, but had significantly more side-effects compared to ramipril monotherapy. ONTARGET is a landmark study, performed according to the highest statistical and clinical standards, providing compelling evidence and clear answers to two important clinical questions.

Afferent nerves in the gut not only signal to the central nervous system but also provide a local efferent-like effect. This effect can modulate intestinal motility and secretion and is postulated to involve the transient receptor potential of the vanilloid type 1 (TRPV1). By using selective TRPV1 agonist and antagonists, we studied the efferent-like effect of afferent nerves in the isolated mouse jejunum. Mouse jejunal muscle strips were mounted in organ baths for isometric tension recordings. Jejunal strips contracted to the TRPV1 agonist capsaicin. Contractions to capsaicin showed rapid tachyphylaxis and were insensitive to tetrodotoxin, hexamethonium, atropine or L-nitroarginine. Capsaicin did not affect contractions to electrical stimulation of enteric motor nerves and carbachol. Tachykinin NK1, NK2 and NK3 receptor blockade by RP67580, nepadutant plus SR-142801 reduced contractions to capsaicin to a similar degree as contractions to substance P. The effect of the TRPV1 antagonists capsazepine, SB-366791, iodo-resiniferatoxin (iodo-RTX) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC) was studied. Capsazepine inhibited contractions not only to capsaicin but also those to carbachol. SB-366791 reduced contractions both to capsaicin and carbachol. Iodo-RTX partially inhibited the contractions to capsaicin without affecting contractions to carbachol. BCTC concentration-dependently inhibited and at the highest concentration used, abolished the contractions to capsaicin without affecting those to carbachol. From these results, we conclude that activation of TRPV1 in the mouse intestine induces a contraction that is mediated by tachykinins most likely released from afferent nerves. The TRPV1-mediated contraction does not involve activation of intrinsic enteric motor nerves. Of the TRPV1 antagonists tested, BCTC combined strong TRPV1 antagonism with TRPV1 selectivity.

G-protein-coupled receptors (GPCRs) respond to external stimuli by activating heterotrimeric G proteins inside the cell. There is increasing evidence that many GPCRs exist as dimers or higher oligomers, but the biochemical nature of such dimers and what roles they have, if any, in signal transduction remains unclear. We conducted a comprehensive study of dimerization of the 5HT2c serotonin receptor using disulphide-trapping experiments. We found a dimer interface between transmembrane (TM) helices IV and V that is markedly sensitive to the state of receptor activation. This dimer seems to be quasisymmetrical in interfacial geometry and asymmetrical in its association with its cognate G alpha protein. We also found a second interface at TM I helices, which is insensitive to the state of activation.

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE-/-) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca2+]i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE-/- and C57BL/6J (WT) mice were evoked by phenylephrine (3x10(-9)-3x10(-5) M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca2+]i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE-/- mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca2+]i in apoE-/- strips with 10(-6) M cyclopiazonic acid or 10(-7) M Ca2+-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE-/- and WT aorta segments. Basal [Ca2+]i was significantly lower in apoE-/- than in WT strips. Relaxation by ACh (3x10(-9)-10(-5) M) was time- and dose-dependently related to [Ca2+]i, but neither ACh-induced relaxation nor Ca2+ mobilization were diminished in apoE-/- strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE-/- mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca2+]i. These findings further point to important differences between basal and stimulated eNOS activity.

Aims: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue., Methods and Results: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production (DeltaT = 0.13 +/- 0.04 degrees C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature., Conclusion: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.

Rats with experimental colitis suffer from impaired gastric emptying (GE). We previously showed that this phenomenon involves afferent neurons within the pelvic nerve. In this study, we aimed to identify the mediators involved in this afferent hyperactivation. Colitis was induced by trinitrobenzene sulfate (TNBS) instillation. We determined GE, distal front, and geometric center (GC) of intestinal transit 30 min after intragastric administration of a semiliquid Evans blue solution. We evaluated the effects of the transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine (5-10 mg/kg) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 1-10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 microg/kg). To determine TRPV1 receptor antagonist sensitivity, we examined their effect on capsaicin-induced relaxations of isolated gastric fundus muscle strips. Immunocytochemical staining of TRPV1 and RT-PCR analysis of TRPV1 mRNA were performed in dorsal root ganglion (DRG) L6-S1. TNBS-induced colitis reduced GE but had no effect on intestinal motility. Capsazepine reduced GE in controls but had no effect in rats with colitis. At doses that had no effects in controls, BCTC and CGRP-(8-37) significantly improved colitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolished them. TNBS-induced colitis increased TRPV1-like immunoreactivity and TRPV1 mRNA content in pelvic afferent neuronal cell bodies in DRG L6-S1. In conclusion, distal colitis in rats impairs GE via sensitized pelvic afferent neurons. We provided pharmacological, immunocytochemical, and molecular biological evidence that this sensitization is mediated by TRPV1 receptors and involves CGRP release.

The lack of exposure to helminth infections, as a result of improved living standards and medical conditions, may have contributed to the increased incidence of IBD in the developed world. Epidemiological, experimental, and clinical data sustain the idea that helminths could provide protection against IBD. Studies investigating the underlying mechanisms by which helminths might induce such protection have revealed the importance of regulatory pathways, for example, regulatory T-cells. Further investigation on how helminths influence both innate and adaptive immune reactions will shed more light on the complex pathways used by helminths to regulate the hosts immune system. Although therapy with living helminths appears to be effective in several immunological diseases, the disadvantages of a treatment based on living parasites are explicit. Therefore, the identification and characterization of helminth-derived immunomodulatory molecules that contribute to the protective effect could lead to new therapeutic approaches in IBD and other immune diseases.

Acute pancreatitis remains a potentially life-threatening disease associated with gastrointestinal motility disturbances. Prokinetic agents may be useful to overcome these motility disturbances. In this study, we investigated the effect of acute necrotizing pancreatitis (ANP) on gastrointestinal motility in female mice and evaluated the effect of tegaserod, a prokinetic 5-hydroxytryptamine-4 (5HT4) receptor agonist. ANP was induced by feeding mice a choline-deficient ethionine-supplemented diet during 72 h. In vivo intestinal motility was measured as the geometric centre (GC) of 25 glass beads 30-120-360 min after gavage. Colonic peristaltic activity was studied using a modified Trendelenburg set-up. ANP significantly decreased GC 30-120-360 min after bead gavage, associated with a significant increase of myeloperoxidase in the proximal small intestine and colon, but not in the stomach or distal small intestine. Tegaserod significantly ameliorated GC 360 min after bead gavage in control and pancreatitis mice. In isolated colonic segments, ANP significantly decreased the amplitude of peristaltic waves and increased the interval between peristaltic contractions. Tegaserod normalized the disturbed interval. In conclusion, ANP impairs gastric, small intestinal and colonic motility in mice. Tegaserod improves ANP-induced motility disturbances in vivo and in vitro, suggesting a therapeutic benefit of prokinetic 5HT4 receptor agonists in the treatment of pancreatitis-induced ileus.

Background and Purpose: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism., Experimental Approach: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis., Key Results: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin., Conclusions and Implications: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.

Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.