CRISPR/Cas9 screening using unique molecular identifiers

Loss‐of‐function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Algorithmic single‐locus species delimitation: effects of sampling effort,variation and nonmonophyly in four methods and 1870 species of beetles

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode‐based OTU‐picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.     

Body Weight and Health Status: Importance of Socioeconomic Position and Working Conditions

Objective: To examine the association between relative body weight and health status and the potential modifying effects of socioeconomic position and working conditions on this association. Research Methods and Procedures: The data were derived from three identical cross‐sectional surveys conducted in 2000, 2001, and 2002. Respondents to postal surveys were middle‐aged employees of the City of Helsinki (7148 women and 1799 men, response rate 67%). BMI was based on self‐reported weight and height. Health status was measured by the Short‐Form 36 subscales and component summaries. Results: Body weight was inversely associated with physical health, but in mental health, differences between BMI categories were small and inconsistent. In women, physical health deteriorated monotonically with increasing BMI, whereas in men, poor physical health was found among the obese only. Socioeconomic position did not modify the association between BMI and health. In women, the association between body weight and physical health became stronger with decreasing job control and increasing physical work load, whereas in men, a similar modifying effect was found for high job demands. Discussion: Body weight was associated with physical health only. Lower levels of relative weight in women than in men may be associated with poor physical health. High body weight combined with adverse working conditions may impose a double burden on physical health.     

Take-off analysis of the Olympic ski jumping competition (HS-106 m)

The take-off phase (approximately 6 m) of the jumps of all athletes participating in the individual HS-106 m hill ski jumping competition at the Torino Olympics was filmed with two high-speed cameras. The high altitude of the Pragelato ski jumping venue (1600 m) and slight tail wind in the final jumping round were expected to affect the results of this competition. The most significant correlation with the length of the jump was found in the in-run velocity (r=0.628, p<0.001, n=50). This was a surprise in Olympic level ski jumping, and suggests that good jumpers simply had smaller friction between their skis and the in-run tracks and/or the aerodynamic quality of their in-run position was better. Angular velocity of the hip joint of the best jumpers was also correlated with jumping distance (r=0.651, p<0.05, n=10). The best jumpers in this competition exhibited very different take-off techniques, but still they jumped approximately the same distance. This certainly improves the interests in ski jumping among athletes and spectators. The comparison between the take-off techniques of the best jumpers showed that even though the more marked upper body movement creates higher air resistance, it does not necessarily result in shorter jumping distance if the exposure time to high air resistance is not too long. A comparison between the first and second round jumps of the same jumpers showed that the final results in this competition were at least partly affected by the wind conditions.     

Alpha-methylated derivatives of 2-arachidonoyl glycerol: synthesis, CB1 receptor activity, and enzymatic stability

Alpha-methylated analogues of the endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), were synthesized aiming to the improved enzymatic stability of 2-AG. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The CB1 receptor activity was determined by the [35S]GTPgammaS binding assay, and the enzymatic stability of alpha-methylated analogues was determined in rat cerebellar membranes. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are clearly more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.     

Ubiquilin 1 modulates amyloid precursor protein trafficking and Abeta secretion

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with alpha-, beta-, or gamma-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (alpha- and beta-forms). Moreover, UBQLN1 knockdown increased levels of secreted Abeta40 and Abeta42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic "gatekeeper" that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Abeta.