In vivo MR imaging of pulmonary perfusion and gas exchange in rats via continuous extracorporeal infusion of hyperpolarized 129Xe

Background

Hyperpolarized (HP) ¹²⁹Xe magnetic resonance imaging (MRI) permits high resolution, regional visualization of pulmonary ventilation. Additionally, its reasonably high solubility (>10%) and large chemical shift range (>200 ppm) in tissues allow HP ¹²⁹Xe to serve as a regional probe of pulmonary perfusion and gas transport, when introduced directly into the vasculature. In earlier work, vascular delivery was accomplished in rats by first dissolving HP ¹²⁹Xe in a biologically compatible carrier solution, injecting the solution into the vasculature, and then detecting HP ¹²⁹Xe as it emerged into the alveolar airspaces. Although easily implemented, this approach was constrained by the tolerable injection volume and the duration of the HP ¹²⁹Xe signal.

Methods and Principal Findings

Here, we overcome the volume and temporal constraints imposed by injection, by using hydrophobic, microporous, gas-exchange membranes to directly and continuously infuse ¹²⁹Xe into the arterial blood of live rats with an extracorporeal (EC) circuit. The resulting gas-phase ¹²⁹Xe signal is sufficient to generate diffusive gas exchange- and pulmonary perfusion-dependent, 3D MR images with a nominal resolution of 2×2×2 mm³. We also show that the ¹²⁹Xe signal dynamics during EC infusion are well described by an analytical model that incorporates both mass transport into the blood and longitudinal relaxation.

Conclusions

Extracorporeal infusion of HP ¹²⁹Xe enables rapid, 3D MR imaging of rat lungs and, when combined with ventilation imaging, will permit spatially resolved studies of the ventilation-perfusion ratio in small animals. Moreover, EC infusion should allow ¹²⁹Xe to be delivered elsewhere in the body and make possible functional and molecular imaging approaches that are currently not feasible using inhaled HP ¹²⁹Xe.     

Exploring Sequence Characteristics Related to High-Level Production of Secreted Proteins in Aspergillus niger

Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.     

Why does the immune system of Atlantic cod lack MHC II?

MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).     

Periconceptional Maternal Folic Acid Use of 400 μg per Day Is Related to Increased Methylation of the IGF2 Gene in the Very Young Child

Background

Countries worldwide recommend women planning pregnancy to use daily 400 µg of synthetic folic acid in the periconceptional period to prevent birth defects in children. The underlying mechanisms of this preventive effect are not clear, however, epigenetic modulation of growth processes by folic acid is hypothesized. Here, we investigated whether periconceptional maternal folic acid use and markers of global DNA methylation potential (S-adenosylmethionine and S-adenosylhomocysteine blood levels) in mothers and children affect methylation of the insulin-like growth factor 2 gene differentially methylation region (IGF2 DMR) in the child. Moreover, we tested whether the methylation of the IGF2 DMR was independently associated with birth weight.

Methodology/Principal Findings

IGF2 DMR methylation in 120 children aged 17 months (SD 0.3) of whom 86 mothers had used and 34 had not used folic acid periconceptionally were studied. Methylation was measured of 5 CpG dinucleotides covering the DMR using a mass spectrometry-based method. Children of mother who used folic acid had a 4.5% higher methylation of the IGF2 DMR than children who were not exposed to folic acid (49.5% vs. 47.4%; p = 0.014). IGF2 DMR methylation of the children also was associated with the S-adenosylmethionine blood level of the mother but not of the child (+1.7% methylation per SD S-adenosylmethionine; p = 0.037). Finally, we observed an inverse independent association between IGF2 DMR methylation and birth weight (−1.7% methylation per SD birthweight; p = 0.034).

Conclusions

Periconceptional folic acid use is associated with epigenetic changes in IGF2 in the child that may affect intrauterine programming of growth and development with consequences for health and disease throughout life. These results indicate plasticity of IGF2 methylation by periconceptional folic acid use.     

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.     

Combining bleach and mild predigestion improves ancient DNA recovery from bones

The feasibility of genome‐scale studies from archaeological material remains critically dependent on the ability to access endogenous, authentic DNA. In the majority of cases, this represents a few per cent of the DNA extract, at most. A number of specific pre‐extraction protocols for bone powder aimed to improve ancient DNA recovery before library amplification have recently been developed. Here, we test the effects of combining two of such protocols, a bleach wash and a predigestion step, on 12 bone samples of Atlantic cod and domestic horse aged 750–1350 cal. years before present. Using high‐throughput sequencing, we show that combined together, bleach wash and predigestion consistently yield DNA libraries with higher endogenous content than either of these methods alone. Additionally, the molecular complexity of these libraries is improved and endogenous DNA templates show larger size distributions. Other library characteristics, such as DNA damage profiles or the composition of microbial communities, are little affected by the pre‐extraction protocols. Application of the combined protocol presented in this study will facilitate the genetic analysis of an increasing number of ancient remains and will reduce the cost of whole‐genome sequencing.     

Genome architecture enables local adaptation of Atlantic cod despite high connectivity

Adaptation to local conditions is a fundamental process in evolution; however, mechanisms maintaining local adaptation despite high gene flow are still poorly understood. Marine ecosystems provide a wide array of diverse habitats that frequently promote ecological adaptation even in species characterized by strong levels of gene flow. As one example, populations of the marine fish Atlantic cod (Gadus morhua) are highly connected due to immense dispersal capabilities but nevertheless show local adaptation in several key traits. By combining population genomic analyses based on 12K single nucleotide polymorphisms with larval dispersal patterns inferred using a biophysical ocean model, we show that Atlantic cod individuals residing in sheltered estuarine habitats of Scandinavian fjords mainly belong to offshore oceanic populations with considerable connectivity between these diverse ecosystems. Nevertheless, we also find evidence for discrete fjord populations that are genetically differentiated from offshore populations, indicative of local adaptation, the degree of which appears to be influenced by connectivity. Analyses of the genomic architecture reveal a significant overrepresentation of a large ~5 Mb chromosomal rearrangement in fjord cod, previously proposed to comprise genes critical for the survival at low salinities. This suggests that despite considerable connectivity with offshore populations, local adaptation to fjord environments may be enabled by suppression of recombination in the rearranged region. Our study provides new insights into the potential of local adaptation in high gene flow species within fine geographical scales and highlights the importance of genome architecture in analyses of ecological adaptation.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.