An efficient method to assemble linear DNA templates for in vitro screening and selection systems.

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl. The method is exemplified with the gene coding for a mutant version of O(6)-alkylguanine alkyltransferase, which is efficiently assembled with an N-terminal peptide tag and its 5'- and 3'-untranslated regions that include a T7 promoter, ribosome binding site and T7 terminator. The utility of the method is further corroborated by assembling error-prone PCR libraries and regenerating templates following model affinity selections. This fast and robust method should find widespread application in directed evolution for the assembly of gene libraries and the regeneration of linear DNA templates between successive screening and selection cycles

Wasserstein Gradient Flows for Moreau Envelopes of f-Divergences in Reproducing Kernel Hilbert Spaces

Most commonly used $f$-divergences of measures, e.g., the Kullback-Leibler divergence, are subject to limitations regarding the support of the involved measures. A remedy consists of regularizing the $f$-divergence by a squared maximum mean discrepancy (MMD) associated with a characteristic kernel $K$. In this paper, we use the so-called kernel mean embedding to show that the corresponding regularization can be rewritten as the Moreau envelope of some function in the reproducing kernel Hilbert space associated with $K$. Then, we exploit well-known results on Moreau envelopes in Hilbert spaces to prove properties of the MMD-regularized $f$-divergences and, in particular, their gradients. Subsequently, we use our findings to analyze Wasserstein gradient flows of MMD-regularized $f$-divergences. Finally, we consider Wasserstein gradient flows starting from empirical measures. We provide proof-of-the-concept numerical examples for $f$-divergences with both infinite and finite recession constant.Comment: 46 pages, 13 figure

A DEIM driven reduced basis method for the diffuse Stokes/Darcy model coupled at parametric phase-field interfaces

In this article, we develop a reduced basis method for efficiently solving the coupled Stokes/Darcy equations with parametric internal geometry. To accommodate possible changes in topology, we define the Stokes and Darcy domains implicitly via a phase-field indicator function. In our reduced order model, we approximate the parameter-dependent phase-field function with a discrete empirical interpolation method (DEIM) that enables affine decomposition of the associated linear and bilinear forms. In addition, we introduce a modification of DEIM that leads to non-negativity preserving approximations, thus guaranteeing positive-semidefiniteness of the system matrix. We also present a strategy for determining the required number of DEIM modes for a given number of reduced basis functions. We couple reduced basis functions on neighboring patches to enable the efficient simulation of large-scale problems that consist of repetitive subdomains. We apply our reduced basis framework to efficiently solve the inverse problem of characterizing the subsurface damage state of a complete in-situ leach mining site. © 2022, The Author(s)

A DEIM driven reduced basis method for the diffuse Stokes/Darcy model coupled at parametric phase-field interfaces

In this article, we develop a reduced basis method for efficiently solving the coupled Stokes/Darcy equations with parametric internal geometry. To accommodate possible changes in topology, we define the Stokes and Darcy domains implicitly via a phase-field indicator function. In our reduced order model, we approximate the parameter-dependent phase-field function with a discrete empirical interpolation method (DEIM) that enables affine decomposition of the associated linear and bilinear forms. In addition, we introduce a modification of DEIM that leads to non-negativity preserving approximations, thus guaranteeing positive-semidefiniteness of the system matrix. We also present a strategy for determining the required number of DEIM modes for a given number of reduced basis functions. We couple reduced basis functions on neighboring patches to enable the efficient simulation of large-scale problems that consist of repetitive subdomains. We apply our reduced basis framework to efficiently solve the inverse problem of characterizing the subsurface damage state of a complete in-situ leach mining site. © 2022, The Author(s)

a retrospective cohort study

Background Metastasis of colorectal cancer (CRC) is directly linked to patient survival. We previously identified the novel gene Metastasis Associated in Colon Cancer 1 (MACC1) in CRC and demonstrated its importance as metastasis inducer and prognostic biomarker. Here, we investigate the geographic expression pattern of MACC1 in colorectal adenocarcinoma and tumor buds in correlation with clinicopathological and molecular features for improvement of survival prognosis. Methods We performed geographic MACC1 expression analysis in tumor center, invasive front and tumor buds on whole tissue sections of 187 well-characterized CRCs by immunohistochemistry. MACC1 expression in each geographic zone was analyzed with Mismatch repair (MMR)-status, BRAF/KRAS- mutations and CpG-island methylation. Results MACC1 was significantly overexpressed in tumor tissue as compared to normal mucosa (p < 0.001). Within colorectal adenocarcinomas, a significant increase of MACC1 from tumor center to front (p = 0.0012) was detected. MACC1 was highly overexpressed in 55% tumor budding cells. Independent of geographic location, MACC1 predicted advanced pT and pN-stages, high grade tumor budding, venous and lymphatic invasion (p < 0.05). High MACC1 expression at the invasive front was decisive for prediction of metastasis (p = 0.0223) and poor survival (p = 0.0217). The geographic pattern of MACC1 did not correlate with MMR-status, BRAF/KRAS- mutations or CpG-island methylation. Conclusion MACC1 is differentially expressed in CRC. At the invasive front, MACC1 expression predicts best aggressive clinicopathological features, tumor budding, metastasis formation and poor survival outcome

Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.

In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1 h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3-10 ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification

Towards an advanced observation system for the marine Arctic in the framework of the Pan-Eurasian Experiment (PEEX)

The Arctic marine climate system is changing rapidly, which is seen in the warming of the ocean and atmosphere, decline of sea ice cover, increase in river discharge, acidification of the ocean, and changes in marine ecosystems. Socio-economic activities in the coastal and marine Arctic are simultaneously changing. This calls for the establishment of a marine Arctic component of the Pan-Eurasian Experiment (MA-PEEX). There is a need for more in situ observations on the marine atmosphere, sea ice, and ocean, but increasing the amount of such observations is a pronounced technological and logistical challenge. The SMEAR (Station for Measuring Ecosystem-Atmosphere Relations) concept can be applied in coastal and archipelago stations, but in the Arctic Ocean it will probably be more cost-effective to further develop a strongly distributed marine observation network based on autonomous buoys, moorings, autonomous underwater vehicles (AUVs), and unmanned aerial vehicles (UAVs). These have to be supported by research vessel and aircraft campaigns, as well as various coastal observations, including community-based ones. Major manned drift-ing stations may occasionally be comparable to terrestrial SMEAR flagship stations. To best utilize the observations, atmosphere-ocean reanalyses need to be further developed. To well integrate MA-PEEX with the existing terrestrialatmospheric PEEX, focus is needed on the river discharge and associated fluxes, coastal processes, and atmospheric transports in and out of the marine Arctic. More observations and research are also needed on the specific socioeconomic challenges and opportunities in the marine and coastal Arctic, and on their interaction with changes in the climate and environmental system. MA-PEEX will promote international collaboration; sustainable marine meteorological, sea ice, and oceanographic observations; advanced data management; and multidisciplinary research on the marine Arctic and its interaction with the Eurasian continent.Peer reviewe