Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

Placental 11-Beta Hydroxysteroid Dehydrogenase Methylation Is Associated with Newborn Growth and a Measure of Neurobehavioral Outcome

Background: There is growing evidence that the intrauterine environment can impact the neurodevelopment of the fetus through alterations in the functional epigenome of the placenta. In the placenta, the HSD11B2 gene encoding the 11-beta hydroxysteroid dehydrogenase enzyme, which is responsible for the inactivation of maternal cortisol, is regulated by DNA methylation, and has been shown to be susceptible to stressors from the maternal environment. Methodology/Principal Findings: We examined the association between DNA methylation of the HSD11B2 promoter region in the placenta of 185 healthy newborn infants and infant and maternal characteristics, as well as the association between this epigenetic variability and newborn neurobehavioral outcome assessed with the NICU Network Neurobehavioral Scales. Controlling for confounders, HSD11B2 methylation extent is greatest in infants with the lowest birthweights (P = 0.04), and this increasing methylation was associated with reduced scores of quality of movement (P = 0.04). Conclusions/Significance: These results suggest that factors in the intrauterine environment which contribute to birth outcome may be associated with placental methylation of the HSD11B2 gene and that this epigenetic alteration is in turn associated with a prospectively predictive early neurobehavioral outcome, suggesting in some part a mechanism for th

Effects of a defective ERAD pathway on growth and heterologous protein production in Aspergillus niger

Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation

Treatment outcome of intravenous artesunate in patients with severe malaria in the Netherlands and Belgium

<p>Abstract</p> <p>Background</p> <p>Intravenous (IV) artesunate is the treatment of choice for severe malaria. In Europe, however, no GMP-manufactured product is available and treatment data in European travellers are scarce. Fortunately, artesunate became available in the Netherlands and Belgium through a named patient programme. This is the largest case series of artesunate treated patients with severe malaria in Europe.</p> <p>Methods</p> <p>Hospitalized patients treated with IV artesunate between November 2007 and December 2010 in the Netherlands and Belgium were retrospectively evaluated. Patient characteristics, treatment and clinical outcome were recorded on a standardized form and mortality, parasite clearance times and the occurrence of adverse events were evaluated.</p> <p>Results</p> <p>Of the 68 treated patients, including 55 with severe malaria, two patients died (2/55 = 3.6%). The mean time to 50% parasite clearance (PCT50), 90% and 99% were 4.4 hours (3.9 - 5.2), 14.8 hours (13.0 - 17.2), and 29.5 hours (25.9 - 34.4) respectively. Artesunate was well tolerated. However, an unusual form of haemolytic anaemia was observed in seven patients. The relationship with artesunate remains uncertain.</p> <p>Conclusions</p> <p>Data from the named patient programme demonstrate that IV artesunate is effective and well-tolerated in European travellers lacking immunity. However, increased attention needs to be paid to the possible development of haemolytic anaemia 2-3 weeks after start of treatment.</p> <p>Treatment of IV artesunate should be limited to the period that IV treatment is required and should be followed by a full oral course of an appropriate anti-malarial drug.</p

Differences in Gene Expression between First and Third Trimester Human Placenta: A Microarray Study

BACKGROUND: The human placenta is a rapidly developing organ that undergoes structural and functional changes throughout the pregnancy. Our objectives were to investigate the differences in global gene expression profile, the expression of imprinted genes and the effect of smoking in first and third trimester normal human placentas. MATERIALS AND METHODS: Placental samples were collected from 21 women with uncomplicated pregnancies delivered at term and 16 healthy women undergoing termination of pregnancy at 9-12 weeks gestation. Placental gene expression profile was evaluated by Human Genome Survey Microarray v.2.0 (Applied Biosystems) and real-time polymerase chain reaction. RESULTS: Almost 25% of the genes spotted on the array (n = 7519) were differentially expressed between first and third trimester placentas. Genes regulating biological processes involved in cell proliferation, cell differentiation and angiogenesis were up-regulated in the first trimester; whereas cell surface receptor mediated signal transduction, G-protein mediated signalling, ion transport, neuronal activities and chemosensory perception were up-regulated in the third trimester. Pathway analysis showed that brain and placenta might share common developmental routes. Principal component analysis based on the expression of 17 imprinted genes showed a clear separation of first and third trimester placentas, indicating that epigenetic modifications occur throughout pregnancy. In smokers, a set of genes encoding oxidoreductases were differentially expressed in both trimesters. CONCLUSIONS: Differences in global gene expression profile between first and third trimester human placenta reflect temporal changes in placental structure and function. Epigenetic rearrangements in the human placenta seem to occur across gestation, indicating the importance of environmental influence in the developing feto-placental unit

Diagnostic strategy and timing of intervention in infected necrotizing pancreatitis: an international expert survey and case vignette study

AbstractBackgroundThe optimal diagnostic strategy and timing of intervention in infected necrotizing pancreatitis is subject to debate. We performed a survey on these topics amongst a group of international expert pancreatologists.MethodsAn online survey including case vignettes was sent to 118 international pancreatologists. We evaluated the use and timing of fine needle aspiration (FNA), antibiotics, catheter drainage and (minimally invasive) necrosectomy.ResultsThe response rate was 74% (N = 87). None of the respondents use FNA routinely, 85% selectively and 15% never. Most respondents (87%) use a step-up approach in patients with infected necrosis. Walled-off necrosis (WON) is considered a prerequisite for endoscopic drainage and percutaneous drainage by 66% and 12%, respectively. After diagnosing infected necrosis, 55% routinely postpone invasive interventions, whereas 45% proceed immediately to intervention. Lack of consensus about timing of intervention was apparent on day 14 with proven infected necrosis (58% intervention vs. 42% non-invasive) as well as on day 20 with only clinically suspected infected necrosis (59% intervention vs. 41% non-invasive).DiscussionThe step-up approach is the preferred treatment strategy in infected necrotizing pancreatitis amongst expert pancreatologists. There is no uniformity regarding the use of FNA and timing of intervention in the first 2–3 weeks of infected necrotizing pancreatitis

Barriers to the uptake and use of feedback in the context of summative assessment

Despite calls for feedback to be incorporated in all assessments, a dichotomy exists between formative and summative assessments. When feedback is provided in a summative context, it is not always used effectively by learners. In this study we explored the reasons for this. We conducted individual interviews with 17 students who had recently received web based feedback following a summative assessment. Constant comparative analysis was conducted for recurring themes. The summative assessment culture, with a focus on avoiding failure, was a dominant and negative influence on the use of feedback. Strong emotions were prevalent throughout the period of assessment and feedback, which reinforced the focus on the need to pass, rather than excel. These affective factors were heightened by interactions with others. The influence of prior learning experiences affected expectations about achievement and the need to use feedback. The summative assessment and subsequent feedback appeared disconnected from future clinical workplace learning. Socio-cultural influences and barriers to feedback need to be understood before attempting to provide feedback after all assessments. A move away from the summative assessment culture may be needed in order to maximise the learning potential of assessments

Comparing Pandemic to Seasonal Influenza Mortality: Moderate Impact Overall but High Mortality in Young Children

Background: We assessed the severity of the 2009 influenza pandemic by comparing pandemic mortality to seasonal influenza mortality. However, reported pandemic deaths were laboratory-confirmed - and thus an underestimation - whereas seasonal influenza mortality is often more inclusively estimated. For a valid comparison, our study used the same statistical methodology and data types to estimate pandemic and seasonal influenza mortality. Methods and Findings: We used data on all-cause mortality (1999-2010, 100% coverage, 16.5 million Dutch population) and influenza-like-illness (ILI) incidence (0.8% coverage). Data was aggregated by week and age category. Using generalized estimating equation regression models, we attributed mortality to influenza by associating mortality with ILI-incidence, while adjusting for annual shifts in association. We also adjusted for respiratory syncytial virus, hot/cold weather, other seasonal factors and autocorrelation. For the 2009 pandemic season, we estimated 612 (range 266-958) influenza-attributed deaths; for seasonal influen