Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy

Bikeability and methodological issues using the active commuting route environment scale (ACRES) in a metropolitan setting

<p>Abstract</p> <p>Background</p> <p>Route environments can positively influence people's active commuting and thereby contribute to public health. The Active Commuting Route Environment Scale (ACRES) was developed to study active commuters' perceptions of their route environments. However, bicycle commuters represent a small portion of the population in many cities and thus are difficult to study using population-based material. Therefore, the aim of this study is to expand the state of knowledge concerning the criterion-related validity of the ACRES and the representativity using an advertisement-recruited sample. Furthermore, by comparing commuting route environment profiles of inner urban and suburban areas, we provide a novel basis for understanding the relationship between environment and bikeability.</p> <p>Methods</p> <p>Bicycle commuters from Greater Stockholm, Sweden, advertisement- (n = 1379) and street-recruited (n = 93), responded to the ACRES. Traffic planning and environmental experts from the Municipality of Stockholm (n = 24) responded to a modified version of the ACRES. The criterion-related validity assessments were based on whether or not differences between the inner urban and the suburban route environments, as indicated by the experts and by four existing objective measurements, were reflected by differences in perceptions of these environments. Comparisons of ratings between advertisement- and street-recruited participants were used for the assessments of representativity. Finally, ratings of inner urban and suburban route environments were used to evaluate commuting route environment profiles.</p> <p>Results</p> <p>Differences in ratings of the inner urban and suburban route environments by the advertisement-recruited participants were in accord with the existing objective measurements and corresponded reasonably well with those of the experts. Overall, there was a reasonably good correspondence between the advertisement- and street-recruited participants' ratings. Distinct differences in commuting route environment profiles were noted between the inner urban and suburban areas. Suburban route environments were rated as safer and more stimulating for bicycle-commuting than the inner urban ones. In general, the findings applied to both men and women.</p> <p>Conclusions</p> <p>The overall results show: considerable criterion-related validity of the ACRES; ratings of advertisement-recruited participants mirroring those of street-recruited participants; and a higher degree of bikeability in the suburban commuting route environments than in the inner urban ones.</p

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

The role of the complement system in ischemic stroke and neural plasticity

Evidence from experimental animal studies suggests that complement activation in the brain is a “double-edged sword” as it exerts beneficial or detrimental effects depending on the context. Here, we assessed whether complement activation in the systemic circulation could be a predictive biomarker of functional outcome after stroke. Further, we studied the role of the complement system in brain plasticity and recovery after ischemic stroke. We found that acute and delayed phase plasma levels of C3 and C3a differ substantially among patients suffering from ischemic stroke of different etiology, and the association of plasma C3 and C3a levels with case/control status and with functional outcome is ischemic stroke subtype-dependent. In large vessel disease and cardioembolic stroke patients, C3 levels at 3-month follow up were associated with an unfavorable functional outcome at both 3 months and 2 years after stroke. However, in cardioembolic stroke patients moderate increase in plasma C3a/C3 ratio predicted favorable outcome after 2 years (Paper I and II). Furthermore, two single nucleotide polymorphisms (SNPs) in the C3 gene were found to be associated with ischemic stroke independently of traditional risk factors and one of these SNPs was associated with cryptogenic stroke (Paper III). Also, two SNPs were associated with plasma C3a or C3 levels independently of age, sex and case/control status. Taken together, the role of the complement system in ischemic stroke is strongly dependent on stroke etiology. We have also found that C3a overexpression in mice increased, whereas C3a receptor (C3aR) deficiency decreased the number of post-stroke-born neurons in the peri-infarct cortex without affecting the infarct size. Furthermore, the density of presynaptic puncta and GAP43-positive axonal growth cones in the cortex surrounding the infarct were lower in the C3aR-deficient compared to control mice, while in the C3a-overexpressing mice post stroke axonal plasticity response was increased. Mice lacking C3aR showed a more pronounced sensorimotor functional deficit as assessed by behavioral testing (Paper IV). These results indicate that C3aR signaling should be considered as a target when designing therapeutic strategies to improve functional recovery after ischemic stroke. To study complement-related neural plasticity in a non-pathological context, we performed electrophysiological recordings in the CA1 region of live hippocampal slices of young mice lacking C3 and control mice. We found that the C3-deficient mice had a decreased neurotransmitter release probability but dendritic spine density, and frequency and amplitude of miniature excitatory postsynaptic potentials were comparable in both groups of mice. Behavioral testing using the IntelliCage platform revealed that the C3-deficient mice performed better in the place and reversal learning tasks (Paper V). These findings may have implications for the management of disorders involving synapse elimination, such as Alzheimer’s diseases, autism or multiple sclerosis