Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi

Background: Within the last years, numerous reports described successful application of the CRISPR nucleases Cas9 and Cpf1 for genome editing in filamentous fungi. However, still a lot of efforts are invested to develop and improve protocols for the fungus and genes of interest with respect to applicability, scalability and targeting efficiencies. These efforts are often hampered by the fact that—although many different protocols are available— one have systematically analysed and compared different CRISPR nucleases and different application procedures thereof for the efficiency of single- and multiplex-targeting approaches in the same fungus. Results: We present here data for successful genome editing in the cell factory Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, using the three different nucleases SpCas9, FnCpf1, AsCpf1 guided to four different gene targets of our interest. These included a polyketide synthase (pks4.2), an alkaline protease (alp1), a SNARE protein (snc1) and a potential transcription factor (ptf1). For all four genes, guide RNAs were developed which enabled successful single-targeting and multiplex-targeting. CRISPR nucleases were either delivered to T. thermophilus on plasmids or preassembled with in vitro transcribed gRNA to form ribonucleoproteins (RNPs). We also evaluated the efficiency of single oligonucleotides for site-directed mutagenesis. Finally, we were able to scale down the transformation protocol to microtiter plate format which generated high numbers of positive transformants and will thus pave the way for future high-throughput investigations. Conclusion: We provide here the first comprehensive analysis and evaluation of different CRISPR approaches for a filamentous fungus. All approaches followed enabled successful genome editing in T. thermophilus; however, with different success rates. In addition, we show that the success rate depends on the respective nuclease and on the targeted gene locus. We finally present a practical guidance for experimental considerations aiming to guide the reader for successful implementation of CRISPR technology for other fungi.TU Berlin, Open-Access-Mittel - 201

SMAPH: A Piggyback Approach for Entity-Linking in Web Queries

We study the problem of linking the terms of a web-search query to a semantic representation given by the set of entities (a.k.a. concepts) mentioned in it. We introduce SMAPH, a system that performs this task using the information coming from a web search engine, an approach we call “piggybacking.” We employ search engines to alleviate the noise and irregularities that characterize the language of queries. Snippets returned as search results also provide a context for the query that makes it easier to disambiguate the meaning of the query. From the search results, SMAPH builds a set of candidate entities with high coverage. This set is filtered by linking back the candidate entities to the terms occurring in the input query, ensuring high precision. A greedy disambiguation algorithm performs this filtering; it maximizes the coherence of the solution by itera- tively discovering the pertinent entities mentioned in the query. We propose three versions of SMAPH that outperform state-of-the-art solutions on the known benchmarks and on the GERDAQ dataset, a novel dataset that we have built specifically for this problem via crowd-sourcing and that we make publicly available

A unified approach to thermo-mechano-caloric-characterization of elastocaloric materials

This paper presents a novel approach to characterizing the relevant mechanical, thermal and caloric properties of elastocalorics material in a single testing device. Usually, tensile experiments are performed to determine the rate- and process-depending stress/strain behavior of nickel-titanium-based shape memory alloys and potentially other elastocaloric materials made from metallic alloys. These tests are relevant for, e.g., characterization of hysteresis properties and subsequent calculation of mechanical work input. In addition, simultaneous observation with an infrared camera is useful to understand temperature evolution and maximum temperature changes achievable during the loading/unloading process. Characterization of the caloric properties of the materials determines latent heats and, together with the mechanical work, also the material coefficient of performance. It is typically carried out via differential scanning calorimetry (DSC), which is performed in a separate device and requires a second experiment with different types of samples. Furthermore, DSC measurements do not reflect the way mechanically induced phase transformations trigger the release and absorption of latent heats as it is the case for elastocalorics. In order to provide a more consistent understanding of the relevant elastocaloric material properties, we here present a novel method that (a) allows for a systematic determination of load-dependent latent heats and (b) introduces a comprehensive testing setup and suitable testing routine to determine the mechanical, thermal and caloric parameters in the same experimental device and with the same sample, thus greatly simplifying the overall procedure

Towards Transiently Secure Updates in Asynchronous SDNs

© ACM 2016. This is the author's version of the work. It is posted here for your personal use. Not for redistribution. The definitive Version of Record was published in Proceedings of the 2016 Conference on ACM SIGCOMM 2016 Conference - SIGCOMM ’16, http://dx.doi.org/10.1145/2934872.2959083.Software-Defined Networks (SDNs) promise to overcome the often complex and error-prone operation of tradi- tional computer networks, by enabling programmabil- ity, automation and verifiability. Yet, SDNs also in- troduce new challenges, for example due to the asyn- chronous communication channel between the logically centralized control platform and the switches in the data plane. In particular, the asynchronous commu- nication of network update commands (e.g., OpenFlow FlowMod messages) may lead to transient inconsisten- cies, such as loops or bypassed waypoints (e.g., fire- walls). One approach to ensure transient consistency even in asynchronous environments is to employ smart scheduling algorithms: algorithms which update subsets of switches in each communication round only, where each subset in itself guarantees consistency. In this demo, we show how to change routing policies in a transiently consistent manner. We demonstrate two al- gorithms, namely, Wayup [5] and Peacock [4], which partition the network updates sent from SDN controller towards OpenFlow software switches into multiple rounds as per respective algorithms. Later, the barrier mes- sages are utilized to ensure reliable network updates.EC/FP7/619609/EU/Unifying Cloud and Carrier Networks/UNIF

Data Centric Domain Adaptation for Historical Text with OCR Errors

We propose new methods for in-domain and cross-domain Named Entity Recognition (NER) on historical data for Dutch and French. For the cross-domain case, we address domain shift by integrating unsupervised in-domain data via contextualized string embeddings; and OCR errors by injecting synthetic OCR errors into the source domain and address data centric domain adaptation. We propose a general approach to imitate OCR errors in arbitrary input data. Our cross-domain as well as our in-domain results outperform several strong baselines and establish state-of-the-art results. We publish preprocessed versions of the French and Dutch Europeana NER corpora

Интенсификация возделывания картофеля

В статье описаны требования к качеству посадочного материала. Проведен анализ существующих технологий посадки картофеля. Предложена модернизированная конструкция посадочной машины и соответственно усовершенствованная технология посадки картофеля.The article describes the requirements for the quality of planting material. The analysis of existing potato planting technologies is carried out. A modernized planting machine design and, correspondingly, an improved technology for planting potatoes are proposed

Simultaneous Detection of Longitudinal and Transverse Bunch Signals at a Storage Ring

To understand and control the dynamics in the longitudinal phase space, time-resolved measurements of different bunch parameters are required. For a reconstruction of this phase space, the detector systems have to be synchronized. This reconstruction can be used e.g. for studies of the micro-bunching instability. It occurs if the interaction of the bunch with its own radiation leads to the formation of sub-structures on the longitudinal bunch profile. These sub-structures can grow rapidly -- leading to a sawtooth-like behaviour of the bunch. At KARA, we use a fast-gated intensified camera for energy spread studies, Schottky diodes for coherent synchrotron radiation studies as well as electro-optical spectral decoding for longitudinal bunch profile measurements. For a synchronization, a hardware synchronization scheme is used which compensates for eventual hardware delays. In this paper, the different experimental setups and their synchronization are discussed and first results of synchronous measurements are presented