Industrial Freeform Generation of Microtools by Laser Micro Sintering

Precision tools with structural resolution reaching the 20 micrometer range can be generated on an industrial scale by “laser micro sintering”. Components featuring aspect ratios above 12 and a roughness Ra down to 1.5 micrometers have already been produced from sub micrometer grained metal powders. The components can be generated either firmly attached to a substrate or fixed in an easily separable mode. If supporting structures are employed, undercuts up to 90° are feasible, without, a process parameter dependent maximum angles of undercut below 90° are obtained. The process has been introduced into the market, labeled microSINTERING by 3D-Micromac AG.Mechanical Engineerin

Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

BACKGROUND: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. RESULTS: We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. CONCLUSIONS: Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain

Efficient CRISPR/Cas9-mediated gene knockin in mouse hematopoietic stem and progenitor cells

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells

High-energy spin waves in the spin-1 square-lattice antiferromagnet La2_2NiO4_4

Inelastic neutron scattering is used to study the magnetic excitations of the $S=1$ square-lattice antiferromagnet La$_2$NiO$_4$. We find that the spin waves cannot be described by a simple classical (harmonic) Heisenberg model with only nearest-neighbor interactions. The spin-wave dispersion measured along the antiferromagnetic Brillouin-zone boundary shows a minimum energy at the $(1/2,0)$ position as is observed in some $S=1/2$ square-lattice antiferromagnets. Thus, our results suggest that the quantum dispersion renormalization effects or longer-range exchange interactions observed in cuprates and other $S=1/2$ square-lattice antiferromagnets are also present in La$_2$NiO$_4$. We also find that the overall intensity of the spin-wave excitations is suppressed relative to linear spin-wave theory indicating that covalency is important. Two-magnon scattering is also observed

Subtropical streams harbour higher genus richness and lower abundance of insects compared to boreal streams, but scale matters

Aim: Biological diversity typically varies between climatically different regions, and regions closer to the equator often support higher numbers of taxa than those closer to the poles. However, these trends have been assessed for a few organism groups, and the existing studies have rarely been based on extensive identical surveys in different climatic regions. Location: We conducted standardized surveys of wadeable streams in a boreal (western Finland) and a subtropical (south-eastern Brazil) region, sampling insects identically from 100 streams in each region and measuring the same environmental variables in both regions. Taxon: Aquatic insects. Methods: Comparisons were made at the scales of local stream sites, drainage basins and entire regions. We standardized the spatial extent of the study areas by resampling regional richness based on subsets of sites with similar extents. We examined differences in genus richness and assemblage abundance patterns between the regions using graphical and statistical modelling approaches. Results: We found that while genus accumulation and rank-abundance curves were relatively similar at the regional scale between Finland and Brazil, regional genus richness was higher in the latter but regional abundance much higher in the former region. These regional patterns for richness and abundance were reproduced by basin and local genus richness that were higher in Brazil than in Finland, and assemblage abundance that was much higher in Finland than in Brazil. The magnitude of the difference in genus richness between Brazil and Finland tended to increase from local through basin to regional scales. Main conclusions: Our findings suggest that factors related to evolutionary diversification might explain differences in genus richness between these two climatically different regions, whereas higher nutrient concentrations of stream waters might explain the higher abundance of insects in Finland than in Brazil.Peer reviewe

Diverse soil carbon dynamics expressed at the molecular level

The stability and potential vulnerability of soil organic matter (SOM) to global change remains incompletely understood due to the complex processes involved in its formation and turnover. Here we combine compound-specific radiocarbon analysis with fraction-specific and bulk-level radiocarbon measurements in order to further elucidate controls on SOM dynamics in a temperate and sub-alpine forested ecosystem. Radiocarbon contents of individual organic compounds isolated from the same soil interval generally exhibit greater variation than those among corresponding operationally-defined fractions. Notably, markedly older ages of long-chain plant leaf wax lipids (n-alkanoic acids) imply that they reflect a highly stable carbon pool. Furthermore, marked 14C variations among shorter- and longer-chain n-alkanoic acid homologues suggest that they track different SOM pools. Extremes in SOM dynamics thus manifest themselves within a single compound class. This exploratory study highlights the potential of compound-specific radiocarbon analysis for understanding SOM dynamics in ecosystems potentially vulnerable to global change

Genic and nongenic contributions to natural variation of quantitative traits in maize

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions

Genome-wide discovery and characterization of maize long non-coding RNAs

BACKGROUND: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans- genetic factors. CONCLUSIONS: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping

Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential

Background: Epithelial cell adhesion molecule is overexpressed in bladder tumours and released from bladder cancer cells in vitro. We test the hypotheses that urinary EpCAM could act as a biomarker for primary bladder cancer detection and risk stratification. Methods: Epithelial cell adhesion molecule was measured by ELISA in urine from 607 patients with primary bladder tumours and in urine from 53 non-cancer controls. Mann–Whitney tests and ROC analyses were used to determine statistical significance and discrimination between non-cancer controls and different stages and grades of disease. Multivariable modelling and Kaplan–Meier analyses were used to determine prognostic significance. The structure of urinary EpCAM was investigated by western blotting and mass spectrometry. Results: Urinary EpCAM levels increase with stage and grade of bladder cancer. Alongside grade and stage, elevated urinary EpCAM is an independent indicator of poor prognosis with a hazard ratio of 1.76 for bladder cancer-specific mortality. The soluble form of EpCAM in urine is the extracellular domain generated by cleavage between ala243 and gly244. Further studies are required to define the influence of other urinary tract malignancies and benign urological conditions on urinary EpCAM. Conclusion: The extracellular domain of EpCAM is shed into urine by bladder tumours. Urinary EpCAM is a strong indicator of bladder cancer-specific survival, and may be useful within a multi-marker panel for disease detection or as a stand-alone marker to prioritise the investigation and treatment of patients. The mechanisms and effects of EpCAM cleavage in bladder cancer are worthy of further investigation, and may identify novel therapeutic targets