Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms

Schedulability-Driven Frame Packing for Multi-Cluster Distributed Embedded Systems

We present an approach to frame packing for multi-cluster distributed embedded systems consisting of time-triggered and event-triggered clusters, interconnected via gateways. In our approach, the application messages are packed into frames such that the application is schedulable. Thus, we have also proposed a schedulability analysis for applications consisting of mixed event-triggered and time-triggered processes and messages, and a worst case queuing delay analysis for the gateways, responsible for routing inter-cluster traffic. Optimization heuristics for frame packing aiming at producing a schedulable system have been proposed. Extensive experiments and a real-life example show the efficiency of our frame-packing approach

Selective targeting of activating and inhibitory Smads by distinct WWP2 ubiquitin ligase isoforms differentially modulates TGFβ signalling and EMT

Ubiquitin-dependent mechanisms have emerged as essential regulatory elements controlling cellular levels of Smads and TGFß-dependent biological outputs such as epithelial–mesenchymal transition (EMT). In this study, we identify a HECT E3 ubiquitin ligase known as WWP2 (Full-length WWP2-FL), together with two WWP2 isoforms (N-terminal, WWP2-N; C-terminal WWP2-C), as novel Smad-binding partners. We show that WWP2-FL interacts exclusively with Smad2, Smad3 and Smad7 in the TGFß pathway. Interestingly, the WWP2-N isoform interacts with Smad2 and Smad3, whereas WWP2-C interacts only with Smad7. In addition, WWP2-FL and WWP2-C have a preference for Smad7 based on protein turnover and ubiquitination studies. Unexpectedly, we also find that WWP2-N, which lacks the HECT ubiquitin ligase domain, can also interact with WWP2-FL in a TGFß-regulated manner and activate endogenous WWP2 ubiquitin ligase activity causing degradation of unstimulated Smad2 and Smad3. Consistent with our protein interaction data, overexpression and knockdown approaches reveal that WWP2 isoforms differentially modulate TGFß-dependent transcription and EMT. Finally, we show that selective disruption of WWP2 interactions with inhibitory Smad7 can stabilise Smad7 protein levels and prevent TGFß-induced EMT. Collectively, our data suggest that WWP2-N can stimulate WWP2-FL leading to increased activity against unstimulated Smad2 and Smad3, and that Smad7 is a preferred substrate for WWP2-FL and WWP2-C following prolonged TGFß stimulation. Significantly, this is the first report of an interdependent biological role for distinct HECT E3 ubiquitin ligase isoforms, and highlights an entirely novel regulatory paradigm that selectively limits the level of inhibitory and activating Smads

Molecular understanding of atmospheric particle formation from sulfuric acid and large oxidized organic molecules

Atmospheric aerosols formed by nucleation of vapors affect radiative forcing and therefore climate. However, the underlying mechanisms of nucleation remain unclear, particularly the involvement of organic compounds. Here, we present high-resolution mass spectra of ion clusters observed during new particle formation experiments performed at the Cosmics Leaving Outdoor Droplets chamber at the European Organization for Nuclear Research. The experiments involved sulfuric acid vapor and different stabilizing species, including ammonia and dimethylamine, as well as oxidation products of pinanediol, a surrogate for organic vapors formed from monoterpenes. A striking resemblance is revealed between the mass spectra from the chamber experiments with oxidized organics and ambient data obtained during new particle formation events at the Hyytiälä boreal forest research station. We observe that large oxidized organic compounds, arising from the oxidation of monoterpenes, cluster directly with single sulfuric acid molecules and then form growing clusters of one to three sulfuric acid molecules plus one to four oxidized organics. Most of these organic compounds retain 10 carbon atoms, and some of them are remarkably highly oxidized (oxygen-to-carbon ratios up to 1.2). The average degree of oxygenation of the organic compounds decreases while the clusters are growing. Our measurements therefore connect oxidized organics directly, and in detail, with the very first steps of new particle formation and their growth between 1 and 2 nm in a controlled environment. Thus, they confirm that oxidized organics are involved in both the formation and growth of particles under ambient conditions

Gas phase formation of extremely oxidized pinene reaction products in chamber and ambient air

High molecular weight (300–650 Da) naturally charged negative ions have previously been observed at a boreal forest site in Hyytiälä, Finland. The long-term measurements conducted in this work showed that these ions are observed practically every night between spring and autumn in Hyytiälä. The ambient mass spectral patterns could be reproduced in striking detail during additional measurements of α-pinene (C<sub>10</sub>H<sub>16</sub>) oxidation at low-OH conditions in the Jülich Plant Atmosphere Chamber (JPAC). The ions were identified as clusters of the nitrate ion (NO<sub>3</sub><sup>−</sup>) and α-pinene oxidation products reaching oxygen to carbon ratios of 0.7–1.3, while retaining most of the initial ten carbon atoms. Attributing the ions to clusters instead of single molecules was based on additional observations of the same extremely oxidized organics in clusters with HSO<sub>4</sub><sup>−</sup> (Hyytiälä) and C<sub>3</sub>F<sub>5</sub>O<sub>2</sub><sup>−</sup> (JPAC). The most abundant products in the ion spectra were identified as C<sub>10</sub>H<sub>14</sub>O<sub>7</sub>, C<sub>10</sub>H<sub>14</sub>O<sub>9</sub>, C<sub>10</sub>H<sub>16</sub>O<sub>9</sub>, and C<sub>10</sub>H<sub>14</sub>O<sub>11</sub>. The mechanism responsible for forming these molecules is still not clear, but the initial reaction is most likely ozone attack at the double bond, as the ions are mainly observed under dark conditions. β-pinene also formed highly oxidized products under the same conditions, but less efficiently, and mainly C<sub>9</sub> compounds which were not observed in Hyytiälä, where β-pinene on average is 4–5 times less abundant than α-pinene. Further, to explain the high O/C together with the relatively high H/C, we propose that geminal diols and/or hydroperoxide groups may be important. We estimate that the night-time concentration of the sum of the neutral extremely oxidized products is on the order of 0.1–1 ppt (~10<sup>6</sup>–10<sup>7</sup> molec cm<sup>−3</sup>). This is in a similar range as the amount of gaseous H<sub>2</sub>SO<sub>4</sub> in Hyytiälä during day-time. As these highly oxidized organics are roughly 3 times heavier, likely with extremely low vapor pressures, their role in the initial steps of new aerosol particle formation and growth may be important and needs to be explored in more detail in the future

A Qualitative Comparison of Approaches Supporting Business Process Variability

The increasing adoption of process-aware information systems, together with the reuse of process knowledge, has led to the emergence of process model repositories with large process families, i.e., collections of related process model variants. For managing such related model collections two types of approaches exist. While behavioral approaches take supersets of variants and derive a process variant by hiding and blocking process elements, structural approaches take a base process model as input and derive a process variant by applying a set of change operations to it. However, at the current stage no framework for assessing these approaches exists and it is not yet clear which approach should be better used and under which circumstances. Therefore, to give first insights about this issue, this work compares both approaches in terms of understandability of the produced process model artifacts, which is fundamental for the management of process families and the reuse of their contained process fragments. In addition, the comparison can serve as theoretical basis for conducting experiments as well as for fostering the development of tools managing business process variability

Mobile phone use and risk of acoustic neuroma: results of the Interphone case–control study in five North European countries

There is public concern that use of mobile phones could increase the risk of brain tumours. If such an effect exists, acoustic neuroma would be of particular concern because of the proximity of the acoustic nerve to the handset. We conducted, to a shared protocol, six population-based case–control studies in four Nordic countries and the UK to assess the risk of acoustic neuroma in relation to mobile phone use. Data were collected by personal interview from 678 cases of acoustic neuroma and 3553 controls. The risk of acoustic neuroma in relation to regular mobile phone use in the pooled data set was not raised (odds ratio (OR)=0.9, 95% confidence interval (CI): 0.7–1.1). There was no association of risk with duration of use, lifetime cumulative hours of use or number of calls, for phone use overall or for analogue or digital phones separately. Risk of a tumour on the same side of the head as reported phone use was raised for use for 10 years or longer (OR=1.8, 95% CI: 1.1–3.1). The study suggests that there is no substantial risk of acoustic neuroma in the first decade after starting mobile phone use. However, an increase in risk after longer term use or after a longer lag period could not be ruled out