Impact of <em>UGT1A</em> polymorphisms on inflammation and fibrogenesis in a humanized transgenic mouse model

Fibrogenesis represents a highly dynamic scarring response of the liver resulting from different causes of hepatic injury. A key process capable of activating a broad array of profibrogenic pathways is oxidative stress. Oxidative injury, induced by reactive oxygen species, is a strong inducer of hepatic stellate cell activation which represents a critical event in fibrogenesis. UDP-glucuronosyltransferase 1A (UGT1A) enzymes detoxify a broad range of endo- and xenobiotic compounds thereby contributing to antioxidative effects, modulation of inflammation and cytoprotection. By catalyzing the covalent binding of glucuronic acid to reactive metabolites or xenobiotics, UGT1As generate hydrophilic inactive glucuronides that are more easily eliminated from the body by biliary or renal excretion. Regarding the role of hepatic glucuronidation for the development and avoidance of liver fibrosis only limited data are available. Therefore, the overall objective of the dissertation was to show that the presence of UGT1A polymorphisms leading to impaired transcriptional activation and lower enzymatic UGT1A activity results in aggravated fibrogenesis as a consequence of compromised UGT1A-mediated antioxidative tissue protection. To achieve this goal, fibrosis development was assessed during different models of hepatic injury in humanized transgenic (htg) UGT1A-WT and htgUGT1A-SNP mice (containing a human haplotype of 10 common occurring UGT1A SNPs). The following studies show a considerable effect of UGT1A gene products on fibrosis development and progression, which is found to be dependent on the etiology of liver damage. While lower hepatic UGT1A expression levels correlated with a higher degree of liver fibrosis during bile duct ligation (BDL)-induced obstructive cholestasis, reduced fibrogenesis and hence a protective effect of the low-function SNP variant was observed during hepatic steatosis/steatohepatitis evoked in the experimental setting of high-fat diet-induced liver injury. Coffee consumption is epidemiologically associated with a lower risk of fibrosis progression in humans, and has previously been shown to be a potent inducer of UGT1A gene expression. During BDL, coffee administration increased transcriptional UGT1A activation in both mouse lines accompanied by a significant reduction of liver fibrosis. Moreover, a more pronounced downregulation of fibrosis-related marker genes as well as a potent reduction of oxidative liver injury was detected in htgUGT1A-WT animals. Therefore, a common UGT1A SNP haplotype observed in 10% of the white population, is suggested to represent a considerable risk factor for fibrosis progression in cholestasis-related disease, and could, in part, be functionally compensated by the inductive effects associated with coffee consumption. As a consequence, the beneficial effects of coffee could serve as a potential medical strategy for therapeutically activating UGT1A transcription in the clinical situation of cholestasis. This is the first study experimentally reporting a protective effect of this low-function Gilbert syndrome-associated haplotype leading to milder hepatic steatosis during the development of non-alcoholic steatohepatitis. Although a higher expression of UGT1A enzymes was observed in htgUGT1A-WT mice, htgUGT1A-SNP mice showed lower serum aminotransferase levels, reduced hepatic fat deposition and less fibrosis. The data from these experiments therefore contributes to a better understanding of one of the fastest growing hepatic disorders worldwide by identifying a novel role of UGT1A enzymes during the development of hepatic steatosis. In addition to the antifibrotic effects, coffee intake has further been associated with a reduced risk of hepatocellular carcinoma (HCC) and cirrhosis, a major risk factor for the development of liver cancer in humans. The inadequate processing of carcinogenic xenobiotics, as a consequence of low-activity UGT1A variants, has previously been suggested to contribute or to initiate the process of liver carcinogenesis. Until now, experimental evidence for the evaluation of several case-control studies supporting this hypothesis is missing. This thesis provides experimental data for the protective influence of UGT1A proteins against neoplastic transformation chemically-induced by diethylnitrosamine (DEN). HtgUGT1A-SNP mice, which presumably show a reduced capacity of environmental carcinogen detoxification, exhibit markedly increased nodule incidence and growth, as well as higher aminotransferase activity levels. After coffee + DEN co-treatment, the number of resulting HCC-like tumor lesions decreased in both mouse lines, but was still higher in mice carrying UGT1A SNPs. The reduced UGT1A activity and carcinogen detoxification may therefore represent a relevant risk factor for individual cancer disposition, which can, at least partially, be influenced by coffee consumption. The coffee-mediated activation of human UGT1A genes delivers a potential explanation for the decreased risk for HCC development and other liver diseases in coffee drinkers

In vitro cell compatibility and antibacterial activity of microencapsulated doxycycline designed for improved localized therapy of septic arthritis

OBJECTIVES: For the treatment of septic arthritis in large animals, the local application of antibiotics as a slow release system may be an appropriate means to reach high local bioactivity and low systemic side effects and drug residues. In this study, doxycycline microspheres were developed and tested in vitro for their drug-release properties, suitability for intra-articular application and antimicrobial activity. METHODS: The development of a slow release system was achieved by microencapsulation of the drug into poly(lactide-co-glycolide) microspheres by a novel ultrasonic atomization method. Drug elution was evaluated from microspheres dispersed in elution medium at pre-defined time points by HPLC. Joint-tissue compatibility was tested on cultured bovine synoviocytes by evaluating the expression of pro-inflammatory cytokine mRNA and the production of nitric oxide (NO). Finally, the antimicrobial activity of the released antibiotic was assessed with gram-negative and gram-positive bacteria exposed to release medium sampled at days 1, 7 and 12 after microsphere suspension. RESULTS: An adequate size of the microspheres, sufficient stabilization of doxycycline in aqueous environment and drug release (25 mg microspheres in 4 mL medium) above MIC for bacteria usually isolated in bovine and equine joints were obtained over 15 days. Although the cytokine mRNA expression reflected the excellent tissue compatibility, the results with NO yielded contradictory results. Antimicrobial tests of the release medium proved to match perfectly the activity of non-encapsulated, free doxycycline as reported in the literature. CONCLUSIONS: The newly developed doxycycline delivery system achieved the target specifications and is ready for in vivo testin

Da Vinci robot-assisted radical prostatectomy: initial experience in 50 consecutive cases

Indexación: Web of Science; ScieloObjective: To report our initial experience in 50 cases submitted to a Robotic Radical Prostatectomy (RRP), evaluating results and the learning curve. Material and Methods: From january to october 2010 we performed 50 consecutives cases of RRP with the da Vinci S-HD Surgical System®. The database was performed prospectively, and was analyzed retrospectively. We evaluate demographic data (age, body mass index) and perioperative data such as clinical stage, preoperative PSA (Prostate Specific Antigen), Gleason Score, ASA, operative times, estimated blood loss, morbidity, hospital stay, time of bladder catheterization and positive margins. A statistical analysis of exponential regression was performed to estimate the learning curve. Results: The mean age was 62 years and the most frequent clinical stage was T1c (84%). The mean PSA was 6.36 ng/mL and in 50% of the patients the Gleason Score was 7. The median surgical time was 199 minutes. The mean blood loss was 666 mL (50-4.000 mL). The hospital stay and the average bladder catheterization time were 2 and 6 days, respectively. There were 2 conversions to a laparoscopic approach, none to open surgery, and 8% of postoperative complication (all Clavien 1). Inmediat urinary continence and potency rates were 88.3% and 33.3%, respectively. When comparing the 25 initial cases versus the last 25, there was a decrease in surgical time and estimated blood loss (254 minutes vs 189 minutes and 876 mL vs 467 mL, respectively). We also found a lower rate of positive margins (20% vs 12%). The learning curve statistically estimated is 40 procedures. Conclusion: The surgeon's experience determine a decrease in surgical time, intraoperative bleeding and especially in the rate of positive margins.Objetivo: Comunicar nuestra experiencia inicial en 50 casos de Prostatectomía Radical Robótica (PRR), evaluando resultados y curva de aprendizaje. Material y Métodos: Desde enero a octubre de 2010 se realizaron 50 PRR con el sistema da Vinci S-HD®. La base de datos fue confeccionada en forma prospectiva y se evaluaron en forma retrospectiva los datos demográficos (edad, índice de masa corporal), estadio clínico, valor de Antígeno Prostático Específico (APE), Score de Gleason, ASA, tiempos quirú;rgicos, sangrado estimado, complicaciones, estadía hospitalaria, tiempo de sonda vesical y tasa de márgenes positivos. Se realizó un análisis estadístico de regresión exponencial para estimar la curva de aprendizaje del método. Resultados: La edad media fue de 62 años y el estadio clínico más frecuente fue el T1c (84%). El valor medio de APE fue de 6,36 ng/mL. El score de Gleason en un 50% correspondió al 7 y la media del ASA a 2. La mediana del tiempo quirú;rgico fue de 199 minutos. El sangrado medio estimado fue de 666 mL (50-4.000 mL). La media de la estadía hospitalaria y el tiempo de sonda fueron de 2 y 6 días, respectivamente. Hubo 2 conversiones a cirugía laparoscópica, ninguna a cirugía abierta y un 8% de complicaciones postoperatorias (todas Clavien 1). La tasa de continencia y de potencia inmediata fue de 88,3% y 33,3%, respectivamente. Cuando comparamos los 25 casos iniciales versus los 25 finales hubo un descenso significativo en el tiempo quirú;rgico y sangrado estimado (254 minutos vs 189 minutos y 876 mL vs 467 mL, respectivamente). También encontramos una menor tasa de márgenes positivos en el grupo 2 (12% vs 20%). El análisis estadístico determinó la curva de aprendizaje en 40 procedimientos. Conclusión: Una mayor experiencia del cirujano, determina una disminución en los tiempos quirú;rgicos, sangrado intraoperatorio y sobre todo en la tasa de márgenes positivos.http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-40262011000600011&nrm=is

Ice mass change in Greenland and Antarctica between 1993 and 2013 from satellite gravity measurements

We construct long-term time series of Greenland and Antarctic ice sheet mass change from satellite gravity measurements. A statistical reconstruction approach is developed based on a Principal Component Analysis to combine high-resolution spatial modes from the Gravity Recovery and Climate Experiment (GRACE) mission with the gravity information from conventional satellite track-ing data. Uncertainties of this reconstruction are rigorously assessed; they include temporal limitations for short GRACE measurements, spatial limitations for the low-resolution conventional tracking data measurements, and limitations of the estimated statistical relationships between low and high degree potential coe�cients re ected in the PCA modes. Trends of mass variations in Greenland and Antarctica are assessed against a number of previous studies. The resulting time series for Greenland show a higher rate of mass loss than other methods before 2000, while the Antarctic ice sheet appears heavily in uenced by interannual variations

Application of mechanistic models to separate the effects of mutation, selection, and drift on protein sequence evolution

Mathematical and statistical models are useful for describing and understanding observations in genetics and genomics. These models have to constantly be updated to reflect current biological understanding. As opposed to descriptive and phenomenological models, mechanistic models allow for the extraction of more biologically relevant information based on underlying principles. Mutation, selection, and genetic drift are the three forces guiding evolution. Mechanistic models rooted in population genetics principles allow us to determine how these forces shape observed data. I demonstrate the usage of mechanistic models to relate protein coding sequences to their fitness landscapes and the evolutionary forces shaping them. Using the yeast L. kluyveri, I show the increased cost of protein synthesis due to a large scale introgression with mismatched codon usage. Furthermore, I analyze site- specific selection on amino acids in the beta-lactamase protein TEM, which confers antibiotic resistance in E. coli and related species

Hanslick's deleted ending

We question Mark Evan Bonds’ interpretation of the deleted ending of Eduard Hanslick’s On the Musically Beautiful. We argue that there is no evidence that it reveals a commitment to Pythagoreanism or Idealism. We supply an alternative explanation of the deletion

Emerging Trends in Global Freshwater Availability

Freshwater availability is changing worldwide. Here we quantify 34 trends in terrestrial water storage (TWS) observed by the Gravity Recovery and Climate Experiment (GRACE) satellites during 2002-2016 and categorize their drivers as natural interannual variability, unsustainable groundwater consumption, or climate change. Several of these trends had been lacking thorough investigation and attribution, including massive changes in northwestern China and the Okavango delta. Others are consistent with climate model predictions. This observation-based assessment of how the world's water landscape is responding to human impacts and climate variations provides a blueprint for evaluating and predicting emerging threats to water and food security

Ocean bottom pressure changes lead to a decreasing length-of-day in a warming climate

We use a coupled climate model to evaluate ocean bottom pressure changes in the IPCC-A1B climate scenario. Ocean warming in the 21st and 22nd centuries causes secular oceanic bottom pressure anomalies. The essential feature is a net mass transfer onto shallow shelf areas from the deeper ocean areas, which exhibit negative bottom pressure anomalies. We develop a simple mass redistribution model that explains this mechanism. Regionally, however, distinct patterns of bottom pressure anomalies emerge due to spatially inhomogeneous warming and ocean circulation changes. Most prominently, the Arctic Ocean shelves experience an above-average bottom pressure increase. We find a net transfer of mass from the Southern to the Northern Hemisphere, and a net movement of mass closer towards Earth's axis of rotation. Thus, ocean warming and the ensuing mass redistribution change the length-of-day by -0.12 ms within 200 years, demonstrating that the oceans are capable of exciting nontidal length-of-day changes on decadal and longer timescales