Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

A Gene Transfer Agent and a Dynamic Repertoire of Secretion Systems Hold the Keys to the Explosive Radiation of the Emerging Pathogen Bartonella

Gene transfer agents (GTAs) randomly transfer short fragments of a bacterial genome. A novel putative GTA was recently discovered in the mouse-infecting bacterium Bartonella grahamii. Although GTAs are widespread in phylogenetically diverse bacteria, their role in evolution is largely unknown. Here, we present a comparative analysis of 16 Bartonella genomes ranging from 1.4 to 2.6 Mb in size, including six novel genomes from Bartonella isolated from a cow, two moose, two dogs, and a kangaroo. A phylogenetic tree inferred from 428 orthologous core genes indicates that the deadly human pathogen B. bacilliformis is related to the ruminant-adapted clade, rather than being the earliest diverging species in the genus as previously thought. A gene flux analysis identified 12 genes for a GTA and a phage-derived origin of replication as the most conserved innovations. These are located in a region of a few hundred kb that also contains 8 insertions of gene clusters for type III, IV, and V secretion systems, and genes for putatively secreted molecules such as cholera-like toxins. The phylogenies indicate a recent transfer of seven genes in the virB gene cluster for a type IV secretion system from a cat-adapted B. henselae to a dog-adapted B. vinsonii strain. We show that the B. henselae GTA is functional and can transfer genes in vitro. We suggest that the maintenance of the GTA is driven by selection to increase the likelihood of horizontal gene transfer and argue that this process is beneficial at the population level, by facilitating adaptive evolution of the host-adaptation systems and thereby expansion of the host range size. The process counters gene loss and forces all cells to contribute to the production of the GTA and the secreted molecules. The results advance our understanding of the role that GTAs play for the evolution of bacterial genomes.     

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.     

NMR-based metabonomics of bovine blood: an investigation into the effects of long term storage on plasma samples

Freezers in research institutions often contain a plethora of samples left over from studies performed years or even decades ago. Along with samples stored in biobanks, these could prove to be treasure troves for metabonomic research. Although the influence of sample handling and short to medium term storage on conventionally determined blood parameters has been reported, little is known about the effects of long term storage (years to decades) on plasma samples. The aim of this study was to investigate the influence of long term storage on the metabolite profile and to assess the value of archived samples for metabonomic studies. Heparinised plasma of 22 heifers that had been stored at ?20 °C for between 2 and 15 years was analysed using NMR spectroscopy and statistical analysis techniques. Lactate (principal component 1) explained 79.6 % of variance between all spectra, but was not correlated with storage time. The highest correlation with storage time (R ² = 0.474) was found for betaine, with other metabolites (acetoacetate, histidines, glycerol, lipids and glucose) also showing moderate correlation (R ² values between 0.217 and 0.437). Our results indicate that samples stored for extended periods of time can potentially be used in metabonomics studies, if precautions are taken during data analysis.     

[18F]FLT and [18F]FDG PET for Non-invasive Treatment Monitoring of the Nicotinamide Phosphoribosyltransferase Inhibitor APO866 in Human Xenografts

Introduction

APO866 is a new anti-tumor compound inhibiting nicotinamide phosphoribosyltransferase (NAMPT). APO866 has an anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation in vivo. The aim of this study was non-invasively to study effect of APO866 treatment on [18F]FLT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake.

Methods

In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment. Baseline [18F]FLT or [18F]FDG scans were made before treatment and repeated after 24 hours, 48 hours and 7 days. Tumor volume was followed with computed tomography (CT). Tracer uptake was quantified using small animal PET/CT. One hour after iv injection of tracer, static PET scans were performed. Imaging results were compared with Ki67 immunohistochemistry.

Results

Tumors treated with APO866 had volumes that were 114% (24 h), 128% (48 h) and 130% (Day 7) relative to baseline volumes at Day 0. In the control group tumor volumes were 118% (24 h), 145% (48 h) and 339% (Day 7) relative to baseline volumes Day 0. Tumor volume between the treatment and control group was significantly different at Day 7 (P = 0.001). Compared to baseline, [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. Compared to baseline, [18F]FDG SUVmax was significantly different at Day 7 (P = 0.005) in the APO866 group.

Conclusions

APO866 treatment caused a significant decrease in [18F]FLT uptake 24 and 48 hours after treatment initiation. The early reductions in tumor cell proliferation preceded decrease in tumor volume. The results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies.     

Thermal habitat of adult Atlantic salmon Salmo salar in a warming ocean

The year-round thermal habitat at sea for adult Atlantic salmon Salmo salar (n = 49) from northern Norway was investigated using archival tags over a 10 year study period. During their ocean feeding migration, the fish spent 90% of the time in waters with temperatures from 1.6–8.4°C. Daily mean temperatures ranged from −0.5 to 12.9°C, with daily temperature variation up to 9.6°C. Fish experienced the coldest water during winter (November–March) and the greatest thermal range during the first summer at sea (July–August). Trends in sea-surface temperatures influenced the thermal habitat of salmon during late summer and autumn (August–October), with fish experiencing warmer temperatures in warmer years. This pattern was absent during winter (November–March), when daily mean temperatures ranged from 3.4–5.0°C, in both colder and warmer years. The observations of a constant thermal habitat during winter in both warmer and colder years, may suggest that the ocean distribution of salmon is flexible and that individual migration routes could shift as a response to spatiotemporal alterations of favourable prey fields and ocean temperatures.     

Southern right whales show no behavioral response to low noise levels from a nearby unmanned aerial vehicle

Unmanned aerial vehicles (UAVs) are increasingly used for wildlife research and monitoring, but little information exists on their potential effect on marine mammals. We assessed the effects of a UAV on the behavior of southern right whales (Eubalaena australis) in Australia. Focal follows of ten right whale mother-calf pairs were conducted using a theodolite. Control data were recorded for 30 min, and then a DJI Inspire 1 Pro was flown above the whales for 10 min at 5 m altitude. Potential changes to horizontal behavior (swim speed and turning angle) and surfacing pattern (interbreath intervals) were investigated by comparing mother-calf behavior before and during UAV approaches. Changes in respiration rate were used to quantify energetic effects. We also explored acoustic cue perceptibility of the UAV at 5, 10, and 30 m altitude, by measuring the received UAV underwater noise level on whales equipped with acoustic tags (DTAGs). The received noise levels were 86.0 ± 3.9 dB re 1 μPa, while the measured ambient noise was 80.7 ± 7.3 dB re 1 μPa in the same frequency band (100–1,500 Hz). No behavioral response to the UAV was observed. This provides support for UAVs as a noninvasive tool to study baleen whale behavior and ecophysiology.     

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.     

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.     

Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus

The barnacle Balanus improvisus is the major fouling macroorganism in Swedish waters and it colonizes most man‐made surfaces submerged in the sea. New or impending legislation restricts the use of traditional, hazardous antifouling coatings based on heavy metals, mainly copper and tin. This calls for the development of new non‐toxic methods that prevent barnacle settlement. In this work several adrenoceptor compounds are shown to be very efficient in preventing the settlement of cyprid larvae of B. improvisus. The settlement rate of laboratory‐reared cyprids was studied in hydrophilised polystyrene dishes containing adrenoceptor antagonists and agonists dissolved in seawater. Two of these drugs, medetomidine and clonidine, repeatedly inhibited settlement at concentrations between 1 nM and 10 nM. In the vertebrate adrenoceptor classification system, which separates pharmacological substances according to their receptor affinity, both of these substances are classified as α₂ adrenoceptor agonists. An inhibiting effect on presyn‐aptic receptors is suggested, but the localization of the receptor effect requires futher studies. Experiments also revealed that the inhibiting effect of medetomidine was reversible. Cyprids incubated with medetomidine for 20 h attached and metamorphosed into juvenile barnacles after washing and transferrence to seawater. The antagonizing compound atipamezole reversed the effect of medetomidine. This observation supports the assumption that this substance acts at the receptor level. Studies of the surface affinity of medetomidine revealed a strong tendency to accumulate in solid/ liquid phase boundaries. This ability makes it particularly attractive as a candidate for the development of a slow‐release carrier in marine coatings. Panels coated with medetomidine in an acrylate polymer and exposed in the field reduced the recruitment of B. improvisus by 96% after 4 weeks and by 70% after 8 weeks.