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Supplementary Table S1. GBM-sLCR features

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment.Significance:Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521

Motivation A recent approach to perform genetic tracing of complex biological problems involves the generation of synthetic deoxyribonucleic acid (DNA) probes that specifically mark cells with a phenotype of interest. These synthetic locus control regions (sLCRs), in turn, drive the expression of a reporter gene, such as fluorescent protein. To build functional and specific sLCRs, it is critical to accurately select multiple bona fide cis-regulatory elements from the target cell phenotype cistrome. This selection occurs by maximizing the number and diversity of transcription factors (TFs) within the sLCR, yet the size of the final sLCR should remain limited. Results In this work, we discuss how optimization, in particular integer programing, can be used to systematically address the construction of a specific sLCR and optimize pre-defined properties of the sLCR. Our presented instance of a linear optimization problem maximizes the activation potential of the sLCR such that its size is limited to a pre-defined length and a minimum number of all TFs deemed sufficiently characteristic for the phenotype of interest is covered. We generated an sLCR to trace the mesenchymal glioblastoma program in patients by solving our corresponding linear program with the software optimizer Gurobi. Considering the binding strength of transcription factor binding sites (TFBSs) with their TFs as a proxy for activation potential, the optimized sLCR scores similarly to an sLCR experimentally validated in vivo, and is smaller in size while having the same coverage of TFBSs. Availability and implementation We provide a Python implementation of the presented framework in the Supplementary Material with which an optimal selection of cis-regulatory elements can be calculated once the target set of TFs and their binding strength with their TFBSs is known. Supplementary information Supplementary data are available at Bioinformatics online.

This study presents an application of artificial neural network (ANN) and Bayesian network (BN) for evaluation of jamming risk of the shielded tunnel boring machines (TBMs) in adverse ground conditions such as squeezing grounds. The analysis is based on database of tunneling cases by numerical modeling to evaluate the ground convergence and possibility of machine entrapment. The results of initial numerical analysis were verified in comparison with some case studies. A dataset was established by performing additional numerical modeling of various scenarios based on variation of the most critical parameters affecting shield jamming. This includes compressive strength and deformation modulus of rock mass, tunnel radius, shield length, shield thickness, in situ stresses, depth of over-excavation, and skin friction between shield and rock. Using the dataset, an ANN was trained to predict the contact pressures from a series of ground properties and machine parameters. Furthermore, the continuous and discretized BNs were used to analyze the risk of shield jamming. The results of these two different BN methods are compared to the field observations and summarized in this paper. The developed risk models can estimate the required thrust force in both cases. The BN models can also be used in the cases with incomplete geological and geomechanical properties. Keywords: Bayesian network (BN), Artificial neural network (ANN), Shielded tunnel boring machine (TBM), Jamming risk, Numerical simulation, Squeezing ground

The identification of the most competent embryos for transfer to the uterus constitutes the main challenge of in-vitro fertilization (IVF). We established a metabolomic-based approach applying Fourier Transform Infrared spectroscopy (FTIR) on 130 samples of 3-days embryo culture supernatants from 26 embryos that implanted and 104 that failed. Examining the internal structure of the data by unsupervised multivariate analysis, it was observed that the supernatants of nonimplanted embryos contained highly heterogeneous spectral features. These features were overlapping with metabolic-implantation fingerprints, thus demonstrating that in establishing embryo-assessment models a one-class modelling involving only the samples with positive-implantation outcomes should be applied. Analysis of variance confirmed that the women´s age (>40 years) undermined the implantation of the embryos exhibiting implantation metabolomics, and also that constituted a condition triggering embryos to express nonimplantation metabolomics. We conclude that IVF-success rates can be significantly improved if FTIR spectroscopy is used as an embryo-selection criterion.

Chromatin modulators with antagonistic functions on epithelial-mesenchymal interconversion regulate common pan-cancer genes., Epithelial-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to modulate proliferation, migration, and stress response. Whereas kinase signaling is believed to be an EMT driver, the molecular mechanisms underlying epithelial-mesenchymal interconversion are incompletely understood. Here, we show that the impact of chromatin regulators on EMT interconversion is broader than that of kinases. By combining pharmacological modulation of EMT, synthetic genetic tracing, and CRISPR interference screens, we uncovered a minority of kinases and several chromatin remodelers, writers, and readers governing homeostatic EMT in lung cancer cells. Loss of ARID1A, DOT1L, BRD2, and ZMYND8 had nondeterministic and sometimes opposite consequences on epithelial-mesenchymal interconversion. Together with RNAPII and AP-1, these antagonistic gatekeepers control chromatin of active enhancers, including pan-cancer-EMT signature genes enabling supraclassification of anatomically diverse tumors. Thus, our data uncover general principles underlying transcriptional control of cancer cell plasticity and offer a platform to systematically explore chromatin regulators in tumor-state–specific therapy.

SummaryAltering ubiquitination by disruption of individual deubiquitinating enzymes (DUBs) has proven to affect hematopoietic stem cell (HSC) maintenance. However, comprehensive knowledge of DUB function during hematopoiesis in vivo is lacking. To accomplish this goal, we systematically inactivated DUBs in mouse hematopoietic progenitors using in vivo small hairpin RNAs (shRNAs) screens. We found that multiple DUBs may be individually required for hematopoiesis and that the ubiquitin-specific protease 15 (USP15) is particularly important for the maintenance of murine hematopoietic stem and progenitor cells in vitro and in vivo. Consistently, Usp15 knockout mice exhibited a reduced HSC pool. The defect was intrinsic to HSCs, as demonstrated by competitive repopulation assays. Importantly, USP15 is highly expressed in normal human hematopoietic cells and leukemias, and USP15 depletion in murine early progenitors and myeloid leukemia cells impaired in vitro expansion and increased genotoxic stress. Our study underscores the importance of DUBs in preserving normal hematopoiesis and uncovers USP15 as a critical DUB in safeguarding genome integrity in HSC and in leukemia cells.

Mit der Einfuhrung von Industrie 4.0 werden neue Anforderungen an industrielle Netzwerke gestellt. Eine der neuen Anforderungen ist, deterministische Kommunikation nicht nur in der Maschine, sondern auch Maschinenubergreifend zu ermoglichen. Deterministische Maschinen-Maschinen Kommunikation soll in Zukunft auch Dynamik, Robustheit und hohe Verfugbarkeit unterstutzen. Um herstellerubergreifende deterministische Kommunikation auf Ethernet basierenden Netzwerken umzusetzen, werden Standards benotigt. Die IEEE 802.1 Arbeitsgruppe fur Time-Sensitive-Networking (TSN) hat sich zur Aufgabe gestellt Bausteine fur deterministisches Ethernet fur zeitkritische Applikationen zu standardisieren, welche auch die erweiterten industriellen Anforderungen erfullen. Dieser Beitrag erlautert, wie sich die Bausteine fur klassenbasierte Streams zusammenfugen lassen um flexible, robuste und redundante deterministische Kommunikation zu ermoglichen

Model based feedback control strategies for electrohydraulic braking and active steering systems in passenger cars are considered. Simulation results with a complex vehicle model and measurements from test drives with a real car are shown for brake control. For a μ-split road surface additional drive dynamics control is required like ESP or active front steering (AFS). Therefore, an AFS feedback controller is applied to stabilize the vehicle. Furthermore, the braking control is improved by AFS, assisting the driver to brake in critical situations.

The further improvement of drive dynamic control needs model-based concepts. Two model-based applications are described. Firstly, the one-track model is used to indicate critical driving situations like oversteering or unstable behavior. Experiments with a middle-class vehicle show the results of this supervision and fault detection method. Secondly, it is shown by simulation how the understeering and oversteering behavior can be influenced by anti-roll bar actuation. Then a nonlinear yaw-rate controller for active steering is designed and it is shown by simulation how the steering behavior can be improved by anti-roll actuation.

This paper proposes a control system for active steering of a passenger car based on Local Linear Models. Furthermore, it is investigated how a active suspension system can improve the performance of the active steering system in critical driving situations. Active antiroll bars at the front and rear axle are introduced as active elements in the suspension system. Based on the characteristic velocity of the vehicle a control concept for the antiroll bar stiffness variation at the front and rear axle is proposed. Simulations with a threedimensional vehicle model are carried out to verify the effectiveness of the proposed control system.

From a safety point of view, the braking system is, besides the driver, one of the key subsystems in a car. The driver, as an adaptive control system, might not notice small faults in the hydraulic part of the braking system and sooner or later critical braking situations, e.g. due to a brake-circuit failure, may occur. Most drivers are not capable of dealing with such critical situations. Therefore, this paper investigates the influence of faults in the braking system on the dynamic vehicle behavior and the steering inputs of the driver required to keep the vehicle on the desired course.

Several actinomycetes isolated from nature were able to use both natural rubber (NR) and syntheticcis-1,4-polyisoprene rubber (IR) as a sole source of carbon. According to their degradation behavior, they were divided into two groups. Representatives of the first group grew only in direct contact to the rubber substrate and led to considerable disintegration of the material during cultivation. The second group consisted of weaker rubber decomposers that did not grow adhesively, as indicated by the formation of clear zones (translucent halos) around bacterial colonies after cultivation on NR dispersed in mineral agar. Taxonomic analysis of four selected strains based on 16S rRNA similarity examinations revealed twoGordoniasp. strains, VH2 and Kb2, and oneMycobacterium fortuitumstrain, NF4, belonging to the first group as well as oneMicromonospora aurantiacastrain, W2b, belonging to the second group. Schiff's reagent staining tests performed for each of the strains indicated colonization of the rubber surface, formation of a bacterial biofilm, and occurrence of compounds containing aldehyde groups during cultivation with NR latex gloves. Detailed analysis by means of scanning electron microscopy yielded further evidence for the two different microbial strategies and clarified the colonization efficiency. Thereby, strains VH2, Kb2, and NF4 directly adhered to and merged into the rubber material, while strain W2b produced mycelial corridors, especially on the surface of IR. Fourier transform infrared spectroscopy comprising the attenuated total reflectance technique was applied on NR latex gloves overgrown by cells of theGordoniastrains, which were the strongest rubber decomposers. Spectra demonstrated the decrease in number ofcis-1,4 double bonds, the formation of carbonyl groups, and the change of the overall chemical environment, indicating that an oxidative attack at the double bond is the first metabolic step of the biodegradation process.

Liposomes or oil-in-water emulsions containing egg yolk phosphatidylcholine (EPC) were combined with aqueous polyhexamethylene biguanide hydrochloride (PHMB). The bactericidal activity of these preparations against Pseudomonas aeruginosa and Staphylococcus aureus as well as their cytotoxicity on cultured murine fibroblasts (L929 cells) was then assayed for either 30 min or 60 min in the presence of cell culture medium containing 10% fetal bovine serum as surrogate for wound fluid. We used two assay designs: in the first bactericidal activity and cytotoxicity were determined in separate experiments; in the second both were determined in one experiment. Combining PHMB and EPC containing o/w emulsions or liposomes protects mammalian cells without neutralizing the antiseptic effect. From all tested combinations the o/w emulsions containing 0.05% PHMB proved to be superior in this respect to the aqueous preparation.

Sulcus-deepening trochleoplasty restores the trochlear groove in patients with patellofemoral instability and underlying trochlear dysplasia. There are types of dysplasia both with (B and D) and without (A and C) a supratrochlear spur. The aim of this study was to identify influencing factors for the clinical outcome following trochleoplasty.Forty-four knees in 38 patients who underwent trochleoplasty for instability (type A in 9, B in 15, C in 9 and D in 11 knees) were assessed clinically with the Kujala score and radiologically with radiographs and MRI. The median follow-up was 4 (2-7.8) years.At follow-up, the median Kujala score had improved from 68 (29-84) to 90 (42-100) points (P 0.001). Instability (P 0.001) and pain (P = 0.027) decreased significantly, but in 3 knees, pain was worse postoperatively. Twenty-seven knees were ranked as excellent, 10 as good, 2 as fair and 5 as poor. Overall, dysplasia types B and D benefited more from surgery than types A and C. The postoperative MRI revealed no chondrolysis or subchondral necrosis, but deterioration of cartilage on the lateral trochlear facet was identified.Trochleoplasty is a useful and reliable surgical technique to improve patellofemoral instability in patients with a dysplastic trochlea. While improved stability is predictable, pain is less predictable and may even increase following surgery. The overall results were directly dependent on the type of the dysplasia, with a significantly better clinical outcome in type B and D. The clinical relevance of this study is that severe dysplasia can successfully be treated with trochleoplasty.III.

Much effort has been invested in recent years, both observationally and theoretically, to understand the interacting processes taking place in planetary systems consisting of a hot Jupiter orbiting its star within 10 stellar radii. Several independent studies have converged on the same scenario: that a short-period planet can induce activity on the photosphere and upper atmosphere of its host star. The growing body of evidence for such magnetic star-planet interactions includes a diverse array of photometric, spectroscopic and spectropolarimetric studies. The nature of which is modeled to be strongly affected by both the stellar and planetary magnetic fields, possibly influencing the magnetic activity of both bodies, as well as affecting irradiation and non-thermal and dynamical processes. Tidal interactions are responsible for the circularization of the planet orbit, for the synchronization of the planet rotation with the orbital period, and may also synchronize the outer convective envelope of the star with the planet. Studying such star-planet interactions (SPI) aids our understanding of the formation, migration and evolution of hot Jupiters., 8 pages, proceedings of Cool Stars 15, St. Andrews, July 2008, to be published in the Conference Proceedings Series of the American Institute of Physics - "Star-planet interactions" splinter session summary

A classification system based on Fourier transform infrared (FTIR) spectroscopy combined with artificial neural network analysis was designed to differentiate 12 serovars of Listeria monocytogenes using a reference database of 106 well-defined strains. External validation was performed using a test set of another 166 L. monocytogenes strains. The O antigens (serogroup) of 164 strains (98.8%) could be identified correctly, and H antigens were correctly determined in 152 (91.6%) of the test strains. Importantly, 40 out of 41 potentially epidemic serovar 4b strains were unambiguously identified. FTIR analysis is superior to PCR-based systems for serovar differentiation and has potential for the rapid, simultaneous identification of both species and serovar of an unknown Listeria isolate by simply measuring a whole-cell infrared spectrum.

Differentiation of the species within the genus Listeria is important for the food industry but only a few reliable methods are available so far. While a number of studies have used Fourier transform infrared (FTIR) spectroscopy to identify bacteria, the extraction of complex pattern information from the infrared spectra remains difficult. Here, we apply artificial neural network technology (ANN), which is an advanced multivariate data-processing method of pattern analysis, to identify Listeria infrared spectra at the species level. A hierarchical classification system based on ANN analysis for Listeria FTIR spectra was created, based on a comprehensive reference spectral database including 243 well-defined reference strains of Listeria monocytogenes , L. innocua , L. ivanovii , L. seeligeri , and L. welshimeri . In parallel, a univariate FTIR identification model was developed. To evaluate the potentials of these models, a set of 277 isolates of diverse geographical origins, but not included in the reference database, were assembled and used as an independent external validation for species discrimination. Univariate FTIR analysis allowed the correct identification of 85.2% of all strains and of 93% of the L. monocytogenes strains. ANN-based analysis enhanced differentiation success to 96% for all Listeria species, including a success rate of 99.2% for correct L. monocytogenes identification. The identity of the 277-strain test set was also determined with the standard phenotypical API Listeria system. This kit was able to identify 88% of the test isolates and 93% of L. monocytogenes strains. These results demonstrate the high reliability and strong potential of ANN-based FTIR spectrum analysis for identification of the five Listeria species under investigation. Starting from a pure culture, this technique allows the cost-efficient and rapid identification of Listeria species within 25 h and is suitable for use in a routine food microbiological laboratory.

A novel sulphate-reducing bacterium (Al1T) was recovered from a soured oil well in Purdu Bay, Alaska. Light and atomic force microscopy observations revealed that cells were Gram-negative, vibrio-shaped and motile by means of a single polar flagellum. The carbon and energy sources used by the isolate and the salinity, temperature and pH ranges facilitating its growth proved to be typical of a partial lactate-oxidizing, moderately halophilic, mesophilic, sulphate-reducing bacterium. Analysis of the fatty acid profile revealed that C(18 : 0), isoC(15 : 0) and isoC(17 : 1)omega7c were the predominant species. Fatty acid profile and complete 16S rRNA gene sequencing demonstrated the similarity between strain Al1T and members of the genus Desulfovibrio. The position of strain Al1T within the phylogenetic tree indicated that it clustered closely with Desulfovibrio vietnamensis DSM 10520T (98.9 % sequence similarity), a strain recovered from a similar habitat. However, whole-cell protein profiles, Fourier-transform infrared studies and DNA-DNA hybridization demonstrated that, in spite of the high level of 16S rRNA gene sequence similarity, there is sufficient dissimilarity at the DNA sequence level between D. vietnamensis DSM 10520T and strain Al1T (10.2 % similarity) to propose that strain Al1T belongs to a separate species within the genus Desulfovibrio. Based on the results obtained, the name Desulfovibrio alaskensis sp. nov. is therefore proposed, with Al1T (= NCIMB 13491T = DSM 16109T) as the type strain.

Staphylococcus aureus has been well known as a major pathogen for many years, causing a variety of nosocomial infections. The rapid and reliable detection of methicillin-resistant S . aureus (MRSA) organisms is of utmost importance to prevent the spread of infection. Species identification is

The main component of biofilms is water, which can make up to over 90% of the wet weight. Biofilms are therefore considered as hydrogels. Water binding and mobility are crucial for diffusion processes in biofilms as well as for dewatering of sludges. In a FTIR-ATR study, the hydrogen-deuterium exchange in native drinking water mixed population biofilms was investigated non-destructively in a flow-through cell. The exchange process of water is characterized by two steps, the first completed after 49 seconds and the second after 21 minutes, indicating different water retention mechanisms. The process is fully completed after about 2 hours. If the biofilm is treated with chlorine (6 mg L −1 for 12h), exchange happens faster and no steps can be observed, indicating a change in the matrix with respect to water binding properties. It is concluded that the water in the EPS matrix is organized in a fine structure which influences the properties of the biofilm.

Biofouling is a problem in many different industrial fields, causing damage of product or interfering with production processes, ranging from drinking and purified water systems to paper manufacture, heat exchange or cosmetics, pharmaceutical, medical and electronic device industries. Timely countermeasures, optimization and efficacy control depend on monitoring of biofilm growth on surfaces. As water samples give no information about site and extent of biofilms, surface sampling is mandatory. The information about biofilm development should be recorded on line, in real time and non destructively in order to permit the kinetics of deposition or removal to be followed. Three physical methods are presented here: i) a fiber optical device, ii) a differential turbidity measurement device, and iii) an FTIR flow cell. The first two methods are based on light reflectance and detect the deposition of reflecting material. Thus, they are not specific for biofilms but they allow us to detect deposit formation in situ, non destructively and in real time. The third method gives information about the chemical nature of the deposit, allowing us to identify biological material. However, this increase of information requires a significantly higher technical effort.

The investigation of the development and the properties of biofilms is difficult because classical microbiology does not offer non-destructive methods other than microscopical observations. This paper discusses the use of different Fourier transform infrared spectroscopy (FTIR-spectroscopy) techniques as a means to investigate microorganisms in biofilms. FTIR-spectroscopy is suitable for the identification of microorganisms and presents a new addition to taxonomic and genetic methods. The FTIR analysis of bacterial isolates provides fingerprint spectra, allowing the rapid characterization of microbial strains. Secondly, the FTIR-attenuated total reflection (ATR) technique can be used for the observation of biofilms forming directly on the interface of an ATR crystals such as germanium. These crystals can be coated to obtain a surface more relevant to study interfacial processes. Spectra can be acquired non-destructively, in situ and in real time. This method is suitable for fundamental biofilm research, as well as for monitoring of biofilm formation, e.g., in an ultrapure or drinking water systems. Furthermore, FTIR—ATR also allows the rapid analysis of deposits on surfaces, e.g., filtration membranes. The analytical discrimination between microorganisms, inorganic material or other foulants can be obtained. Thirdly, with the diffuse reflectance technique (DRIFT) it is possible to investigate reflecting surfaces like metals or very small samples. The composition of surface coatings like biomass or other surface contaminants can be detected. These different measurement techniques demonstrate that FTIR-spectroscopy is suitable for biofilm and surface analysis and can be applied in many different ways.

Two carbohydrate epitopes were identified by monoclonal antibodies (KCS and E2) and characterized with respect to their immunoreactivity, monosaccharide structure, and location. Immunofluorescence demonstrated the presence of both epitopes on the surfaces of sporocysts, cercariae, and miracidia of Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. However, spatial distribution and density of expression varied among species and developmental stages, and neither epitope was detectable on adult worm surfaces. Both glycans were found in the hemolymph of infected, but not uninfected, intermediate snail hosts. The presence of epitopes in hemolymph, as well as in schistosome eggs, is species-specific for KCS, recognizing only S. mansoni, and partly specific for E2, which reacted predominantly with S. haematobium. Immunoaffinity purification of target antigens for KCS and E2 from hemolymph of infected Biomphalaria and Bulinus, respectively, followed by carbohydrate composition analysis revealed a high content of fucose in both glycans. Methylation analysis demonstrated exclusively terminal fucose for the target antigen of KCS and terminal as well as internal fucose for the one of E2. Removal of terminal fucose abolished reactivity with both monoclonal antibodies. Both glycans are different from previously characterized schistosome carbohydrates. Their biological function(s) remain to be defined.

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. Significance: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses. This article is highlighted in the In This Issue feature, p. 521