Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage.

Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless, a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation, oxidative phosphorylation is essential at all stages of differentiation

Potential of a cyclone prototype spacer to improve in vitro dry powder delivery

Copyright The Author(s) 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPurpose: Low inspiratory force in patients with lung disease is associated with poor deagglomeration and high throat deposition when using dry powder inhalers (DPIs). The potential of two reverse flow cyclone prototypes as spacers for commercial carrierbased DPIs was investigated. Methods: Cyclohaler®, Accuhaler® and Easyhaler® were tested with and without the spacers between 30-60 Lmin-1. Deposition of particles in the next generation impactor and within the devices was determined by high performance liquid chromatography. Results: Reduced induction port deposition of the emitted particles from the cyclones was observed due to the high retention of the drug within the spacers (e.g. salbutamol sulphate (SS): 67.89 ± 6.51 % at 30 Lmin-1 in Cheng 1). Fine particle fractions of aerosol as emitted from the cyclones were substantially higher than the DPIs alone. Moreover, the aerodynamic diameters of particles emitted from the cyclones were halved compared to the DPIs alone (e.g. SS from the Cyclohaler® at 4 kPa: 1.08 ± 0.05 μm vs. 3.00 ± 0.12 μm, with and without Cheng 2, respectively) and unaltered with increased flow rates. Conclusion: This work has shown the potential of employing a cyclone spacer for commercial carrier-based DPIs to improve inhaled drug delivery.Peer reviewe

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production. Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group I was orally infected with 1000 +/- 50 embryonated A. galli eggs. Group 2 received 10(4) cfu p. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for I I weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded. The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion. In conclusion a negative interaction between A. galli and R multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli

Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

<p>Abstract</p> <p>Background</p> <p>During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.</p> <p>Results</p> <p>Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).</p> <p>Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.</p> <p>Conclusions</p> <p>The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.</p

Current guidelines for the management of asthma in young children

The diagnosis and management of asthma in young children is difficult, since there are many different wheezy phenotypes with varying underlying aetiologies and outcomes. This review discusses the different approaches to managing young children with wheezy illnesses presented in recently published global guidelines. Four major guidelines published since 2007 are considered. Helpful approaches are presented to assist the clinician to decide whether a clinical diagnosis of asthma can, or should be made in a young child with a recurrent wheezy illness and which treatments would be appropriate, dependent on risk factors, age of presentation, response to initial treatment and safety considerations. Each of the guidelines provide useful information for clinicians assessing young children with recurrent wheezy illnesses. There are differences in classification of the disease and treatment protocols. Although a firm diagnosis of asthma may only be made retrospectively in some cases and there are several effective guidelines to initiating treatment. Consistent review of the need for ongoing treatment with a particular pharmacological modality is essential, since many children with recurrent wheezing in infancy go into spontaneous remission. It is probable that newer biomarkers of airway inflammation will assist the clinician as to when to initiate and when to continue pharmacological treatment in the future

Novel de novo BRCA2 mutation in a patient with a family history of breast cancer

<p>Abstract</p> <p>Background</p> <p><it>BRCA2 </it>germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel <it>de novo BRCA2 </it>splice site mutation found in a patient exhibiting a ductal carcinoma at the age of 40.</p> <p>Methods</p> <p>Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the <it>BRCA1 </it>and <it>BRCA2 </it>genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from whole blood. The paternity was determined by single nucleotide polymorphism (SNP) microarray analysis. Parental origin of the <it>de novo </it>mutation was determined by establishing mutation-SNP haplotypes by variant specific PCR, while <it>de novo </it>and mosaic status was investigated by sequencing of DNA from leucocytes and carcinoma tissue.</p> <p>Results</p> <p>A novel <it>BRCA2 </it>variant in the splice donor site of exon 21 (nucleotide 8982+1 G→A/c.8754+1 G→A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon and synthesis of a truncated BRCA2 protein. The aberrant splicing was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. The mutation was not found in any of the patient's parents or in the mother's carcinoma, showing it is a <it>de novo </it>mutation. Variant specific PCR indicates that the mutation arose in the male germ-line.</p> <p>Conclusion</p> <p>We conclude that the novel <it>BRCA2 </it>splice variant is a <it>de novo </it>mutation introduced in the male spermatozoa that can be classified as a disease causing mutation.</p