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In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life., eLife, 11, ISSN:2050-084X

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development., Nature Methods, 19 (1), ISSN:1548-7105, ISSN:1548-7091

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed ‘positional identity’. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Conversion/alloy active materials, such as ZnO, are one of the most promising candidates to replace graphite anodes in lithium-ion batteries. Besides a high specific capacity (qZnO = 987 mAh g-1), ZnO offers a high lithium-ion diffusion and fast reaction kinetics, leading to a high-rate capability, which is required for the intended fast charging of battery electric vehicles. However, lithium-ion storage in ZnO is accompanied by the formation of lithium-rich solid electrolyte interphase (SEI) layers, immense volume expansion, and a large voltage hysteresis. Nonetheless, ZnO is appealing as an anode material for lithium-ion batteries and is investigated intensively. Surprisingly, the conclusions reported on the reaction mechanism are contradictory and the formation and composition of the SEI are addressed in only a few works. In this work, we investigate lithiation, delithiation, and SEI formation with ZnO in ether-based electrolytes for the first time reported in the literature. The combination of operando and ex situ experiments (cyclic voltammetry, X-ray photoelectron spectroscopy, X-ray diffraction, coupled gas chromatography and mass spectrometry, differential electrochemical mass spectrometry, and scanning electron microscopy) clarifies the misunderstanding of the reaction mechanism. We evidence that the conversion and alloy reaction take place simultaneously inside the bulk of the electrode. Furthermore, we show that a two-layered SEI is formed on the surface. The SEI is decomposed reversibly upon cycling. In the end, we address the issue of the volume expansion and associated capacity fading by incorporating ZnO into a mesoporous carbon network. This approach reduces the capacity fading and yields cells with a specific capacity of above 500 mAh g-1 after 150 cycles.

Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR–Cas9 system. The protocol, which takes 7–8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)–CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome. This protocol describes strategies for targeted genome modification in axolotls. Eggs are injected with a CAS9–gRNA ribonucleoprotein complex, which allows for efficient generation of knockout and knock-in animals and immediate phenotypic analysis.

Catholyte in all-vanadium redox-flow battery (VRFB) which consists of vanadium salts dissolved in sulphuric acid is known to be stabilized by phosphoric acid to slow down the thermal aging at temperatures higher than 40 degrees C. To reveal the role of phosphoric acid, the thermally-induced aggregation is investigated using variable-temperature V-51, P-31, O-17, H-1 nuclear magnetic resonance (NMR) spectroscopy and dynamic light scattering (DLS). The results indicate that the thermal stabilization of vanadium(V) electrolyte is attained by the involvement of monomeric and dimeric vanadium(V) species in the reaction with phosphoric acid which is concurrent to the formation of neutral hydroxo-aqua vanadium(V) precipitation precursor. The dimers are stabilized by counter ions due to association reaction or if such stabilization is not possible, precipitation of vanadium pentoxide is favored. The evolution of particles size distributions at 50 degrees C in electrolyte samples containing 1.6 M vanadium and 4.0 M total sulphate and the pathways of precipitate formation are discussed. The optimal total phosphate concentration is found to be of 0.15 M. However, the induction time is assumed to be dependent not only on the total phosphate concentrations, but also on the ratio of total vanadium(V) to sulphate concentrations.

Wie Unternehmen funktionieren, lernen Kinder und Jugendliche am besten, wenn sie ein solches fuhren. Das Bildungsprojekt „School2Start-up – Schuler entdecken Wirtschaft“ der Wirtschaftsinitiative Wissensfabrik ermoglicht genau das: Schuler grunden ihre eigene Firma. Sie verwandeln ihr Klassenzimmer in ein Buro oder eine Produktionsstatte und lernen, wie sie ein Unternehmen aufbauen und (erfolgreich) fuhren. Begleitet werden sie dabei von einem Paten aus einem Unternehmen des Wissensfabrik-Netzwerks. Das Unternehmen unterstutzt die Lehrkrafte und steht den „Jungunternehmern“ als Experte zur Seite. School2Start-up begleitet die Jugendlichen auf ihrem Weg vom Finden einer Geschaftsidee uber das Erstellen eines Geschaftsplans und dessen Umsetzung bis zur Bilanzierung und Reflexion. Denn: Die so gewonnenen Erfahrungen sind letztlich auch fur die berufliche Orientierung der Schulerinnen und Schuler sowie das Nachdenken uber Nachhaltigkeit wichtig.

// Tobias Gerber 1, * , Edith Willscher 2, * , Henry Loeffler-Wirth 2 , Lydia Hopp 2 , Dirk Schadendorf 3 , Manfred Schartl 4, 5, 6 , Ulf Anderegg 7 , Gray Camp 1 , Barbara Treutlein 1 , Hans Binder 2 , Manfred Kunz 7 1 Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany 2 Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany 3 Department of Dermatology, Venereology and Allergology, University Hospital Essen, 45147 Essen, Germany 4 Department of Physiological Chemistry, University of Wurzburg, Biozentrum, Am Hubland, 97074 Wurzburg, Germany 5 Comprehensive Cancer Center Mainfranken, University Clinic Wurzburg, 97080 Wurzburg, Germany 6 Institute for Advanced Study, 3572 Texas A&M University, College Station, Texas 77843-3572, USA 7 Department of Dermatology, Venereology and Allergology, University of Leipzig, 04103 Leipzig, Germany * These authors contributed equally to this work Correspondence to: Manfred Kunz, email: manfred.kunz@medizin.uni-leipzig.de Keywords: melanoma, single cell transcriptome sequencing, stem cells Received: May 18, 2016 Accepted: November 12, 2016 Published: November 26, 2016 ABSTRACT Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/ NRAS wild type and BRAF wild type/ NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/ NRAS wild type and BRAF wild type/ NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.

Summary Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration., Graphical abstract, Highlights • Fibroblast-derived Prrx1+ cells are the main constituent of a frog limb blastema • Frog fibroblasts only undergo partial dedifferentiation due to intrinsic limitations • Adult chondrogenesis is distinct from the embryonic program, Lin et al. systematically compared the response to limb amputation in axolotls and African clawed frogs. Unlike in axolotls, frog limb fibroblasts are incapable of fully re-activating developmental programs required for complete limb regeneration. This work defines intrinsic restrictions in fibroblasts as a limitation to adult regeneration.

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates. ISSN:1097-6256 ISSN:1546-1726

Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Today’s global competition and rising prizes for resources force manufacturing companies to integrate and make use of various IT systems in their production environment. Those IT systems need to be directly connected with the manufacturing plants to optimize the value added chain in all levels of the enterprise. The connection, that should be flexible, needs to be done in increasingly shorter time, resulting in fixed communication structures. Such inadequate connection of the business (strategic) and technical (operation) levels increases the planning and implementation effort dramatically due to shortened reconfiguration tasks. This paper examines a flexible communication architecture approach for a vertical integration of production process-relevant data, i.e., for closing the gap between the business and technical levels. The approach enables the transfer of information in form of key performance indicators. The key performance indicators will support decision-making processes in the manufacturing companies. To prove the feasibility of the vertical integration approach a prototype is implemented and evaluated in the SmartFactory KL.

This paper deals with a browser based approach of multi-reality interfaces in the context of e-maintenance in modular factory environments, specifically remote monitoring and maintenance scenarios. Through their rich visualization capabilities (stereoscopic video & 3D graphics) multi-reality interfaces offer a way to aggregate and structure the wide-spread variety of information from different sources that is required to analyze such complex scenarios. Additionally, they serve the purpose of making collaboration with on-site personal easier and more efficient in time and cost. Finally, the browser based implementation fits very well into the web based world that a modular factory environment represents and make the presented solution extremely adaptive to (non-)industrial HMI.

In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper

Zu Unternehmenssoftware gehören Systeme, die technische Prozesse innerhalb eines Unternehmens steuern, aber auch Anwendungen, welche die gesamte Wertschöpfungskette abbilden. Dafür sind Informations- und eingebettete Systeme zu integrieren - innerhalb eines Unternehmens und zwischen verschiedenen Unternehmen.

DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

To fulfill increasing requirements in the manufacturing sector, companies are facing several challenges. Three major challenges have been identified regarding time-to-market, vertical feedback loops, and level of automation. Grafchart, a graphical language aimed for supervisory control applications, can be used from the process-planning phase, through the implementation phase and all the way to the phase for execution of the process control logics, on the lower levels of the automation triangle along the life cycle axis. This work in progress examines that the same process-based engineering approach can be used on the higher levels of the automation triangle along the enterprise axis interconnecting both axes. By splitting the execution engine and the visualization engine of Grafchart various different visualization tools could potentially be used, however connected by the shared Grafchart semantics. Traditional Business languages (e.g. BPMN) could therefore continue to be used for the process-planning phase whereas traditional production languages (e.g. Grafchart or other sequential function charts-like languages) could be used for the execution. Since they are connected through the semantics, advantages regarding the three identified challenges could be achieved: time-to-market could be reduced, the time delays in the vertical feedback loops could be reduced by Key Performance Indicator visualization and eventing, and the level of automation could be increased.

The growing competition between manufacturers demands a higher versatility of factory automation technology. Component based automation principles propose an opportunity to address these challenges by encapsulating the functionality of mechatronic components in an abstract way. Service-oriented approaches depict a promising possibility to realize such architectures. Service discovery and orchestration are the key functionalities of such systems. However, standard web service technologies alone are not suited for the creation of highly-flexible automation systems. We describe a semantic service discovery and orchestration system, which is based on different semantic service technologies, and provide a concept towards the creation of adaptive production processes based on our experiences during practical implementation.

Diverse regions develop within cerebral organoids generated from human induced pluripotent stem cells (iPSCs), however it has been a challenge to understand the lineage dynamics associated with brain regionalization. Here we establish an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during cerebral organoid development. We infer fate-mapped whole organoid phylogenies over a scarring time course, and reconstruct progenitor-neuron lineage trees within microdissected cerebral organoid regions. We observe increased fate restriction over time, and find that iPSC clones used to initiate organoids tend to accumulate in distinct brain regions. We use lineage-coupled spatial transcriptomics to resolve lineage locations as well as confirm clonal enrichment in distinctly patterned brain regions. Using long term 4-D light sheet microscopy to temporally track nuclei in developing cerebral organoids, we link brain region clone enrichment to positions in the neuroectoderm, followed by local proliferation with limited migration during neuroepithelial formation. Our data sheds light on how lineages are established during brain organoid regionalization, and our techniques can be adapted in any iPSC-derived cell culture system to dissect lineage alterations during perturbation or in patient-specific models of disease.