Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

Nonparametric Modeling of Dynamic Functional Connectivity in fMRI Data

Dynamic functional connectivity (FC) has in recent years become a topic of interest in the neuroimaging community. Several models and methods exist for both functional magnetic resonance imaging (fMRI) and electroencephalography (EEG), and the results point towards the conclusion that FC exhibits dynamic changes. The existing approaches modeling dynamic connectivity have primarily been based on time-windowing the data and k-means clustering. We propose a non-parametric generative model for dynamic FC in fMRI that does not rely on specifying window lengths and number of dynamic states. Rooted in Bayesian statistical modeling we use the predictive likelihood to investigate if the model can discriminate between a motor task and rest both within and across subjects. We further investigate what drives dynamic states using the model on the entire data collated across subjects and task/rest. We find that the number of states extracted are driven by subject variability and preprocessing differences while the individual states are almost purely defined by either task or rest. This questions how we in general interpret dynamic FC and points to the need for more research on what drives dynamic FC.Comment: 8 pages, 1 figure. Presented at the Machine Learning and Interpretation in Neuroimaging Workshop (MLINI-2015), 2015 (arXiv:1605.04435

Fetal Programming of the Endocrine Pancreas:Impact of a Maternal Low-Protein Diet on Gene Expression in the Perinatal Rat Pancreas

In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy

Major coastal impact induced by a 1000-year storm event

Extreme storms and storm surges may induce major changes along sandy barrier coastlines, potentially causing substantial environmental and economic damage. We show that the most destructive storm (the 1634 AD storm) documented for the northern Wadden Sea within the last thousand years both caused permanent barrier breaching and initiated accumulation of up to several metres of marine sand. An aggradational storm shoal and a prograding shoreface sand unit having thicknesses of up to 8 m and 5 m respectively were deposited as a result of the storm and during the subsequent 30 to 40 years long healing phase, on the eroded shoreface. Our results demonstrate that millennial-scale storms can induce large-scale and long-term changes on barrier coastlines and shorefaces, and that coastal changes assumed to take place over centuries or even millennia may occur in association with and be triggered by a single extreme storm event

USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products

We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications