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Over the past decade, terahertz spectroscopy has evolved into a versatile tool for chemically selective sensing and imaging applications. In particular, the potential to coherently generate and detect short, and hence, broadband terahertz pulses led to the development of efficient and compact spectrometers for this interesting part of the electromagnetic spectrum, where common packaging materials are transparent and many chemical compounds show characteristic absorptions. Although early proof-of-principle demonstrations have shown the great potential of terahertz spectroscopy for sensing and imaging, the technology still often lacks the required sensitivity and suffers from its intrinsically poor spatial resolution. In this review we discuss the current potential of terahertz pulse spectroscopy and highlight recent technological advances geared towards both enhancing spectral sensitivity and increasing spatial resolution. [graphic removed]

Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis 1 , which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles 2 – 6 . Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react ‘on demand’ to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness. Sequencing of total and ribosome-associated mRNAs enables identification of specific mRNAs that are differentially translationally regulated along the neuronal stem cell lineage, independently of their availability.

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5 , while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them. The heterogeneity underlying cancer organoid phenotypes is not yet well understood. Here, the authors develop an imaging analysis assay for high throughput phenotypic screening of colorectal organoids that allows to define specific morphological changes that occur following different drug treatments.

Using conventional materials, the resolution of focusing and imaging devices is limited by diffraction to about half the wavelength of light, as high spatial frequencies do not propagate in isotropic materials. Wire array metamaterials, because of their extreme anisotropy, can beat this limit; however, focusing with these has only been demonstrated up to microwave frequencies and using propagation over a few wavelengths only. Here we show that the principle can be scaled to frequencies orders of magnitudes higher and to considerably longer propagation lengths. We demonstrate imaging through straight and tapered wire arrays operating in the terahertz spectrum, with unprecedented propagation of near field information over hundreds of wavelengths and focusing down to 1/28 of the wavelength with a net increase in power density. Applications could include in vivo terahertz-endoscopes with resolution compatible with imaging individual cells. Wire array metamaterials can be used to beat the fundamental diffraction limit of light, but most demonstrations have been limited to microwaves and very short propagation lengths. Tuniz et al . scale these metamaterial fibres up to the terahertz region over longer distances and show focussing down to λ /28.

The maternal-to-zygotic transition (MZT) is a process that occurs in animal embryos at the earliest developmental stages, during which maternally deposited mRNAs and other molecules are degraded and replaced by products of the zygotic genome. The zygotic genome is not activated immediately upon fertilization, and in the pre-MZT embryo post-transcriptional control by RNA-binding proteins (RBPs) orchestrates the first steps of development. To identify relevant Drosophila RBPs organism-wide, we refined the RNA interactome capture method for comparative analysis of the pre- and post-MZT embryos. We determine 523 proteins as high-confidence RBPs, half of which were not previously reported to bind RNA. Comparison of the RNA interactomes of pre- and post-MZT embryos reveals high dynamicity of the RNA-bound proteome during early development, and suggests active regulation of RNA binding of some RBPs. This resource provides unprecedented insight into the system of RBPs that govern the earliest steps of Drosophila development. Early development is controlled by maternally deposited mRNAs and the RNA-binding proteins (RBPs) that regulate them. Here the authors describe the identification of a large number of RBPs bound to polyadenylated RNAs in Drosophila embryos before and after the maternal-to-zygotic transition, revealing changes in RBPs activity during development.

RNAi screening combined with automated microscopy of Drosophila melanogaste r cells reveal genetic interactions and allow the reconstruction of signaling pathways. The analysis of synthetic genetic interaction networks can reveal how biological systems achieve a high level of complexity with a limited repertoire of components. Studies in yeast and bacteria have taken advantage of collections of deletion strains to construct matrices of quantitative interaction profiles and infer gene function. Yet comparable approaches in higher organisms have been difficult to implement in a robust manner. Here we report a method to identify genetic interactions in tissue culture cells through RNAi. By performing more than 70,000 pairwise perturbations of signaling factors, we identified >600 interactions affecting different quantitative phenotypes of Drosophila melanogaster cells. Computational analysis of this interaction matrix allowed us to reconstruct signaling pathways and identify a conserved regulator of Ras-MAPK signaling. Large-scale genetic interaction mapping by RNAi is a versatile, scalable approach for revealing gene function and the connectivity of cellular networks.

Incorporation of time information into the annotation of distinct biological states in automated fluorescence time-lapse live-cell imaging of complex cellular dynamics reduces both classification noise and confusion between cell states with similar morphology. A computational framework for achieving this is implemented in the open-source software package CellCognition. Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging–based screening with assays that directly score cellular dynamics.

Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.

DEHP is a plasticizer which has been used in plastic products of everyday use for decades. Studies in mice and murine cell culture models identified DEHP as an endocrine disruptor that may also act as an obesogen. As this is of high concern in respect of the worldwide obesity epidemic, our aim is the translation of these findings into a human model system. On the basis of DOHaD, we investigated the influence of an environmentally relevant dose of DEHP [50 µg/ml] on adipogenesis in the human cell culture model SGBS. Pre-adipocytes were exposed to DEHP and differentiated into mature adipocytes. At different stages of differentiation, markers of adipogenesis like GLUT4, FABP4, LPL and PPARs, and of signaling pathways like AMPK/ACC2, JAK/STAT and MAPK were analyzed. Functional markers like adipokine secretion and triglyceride content as well as ROS production were measured in mature adipocytes. We found significantly lower expression levels of adipogenic markers, a reduction in lipid accumulation, higher leptin- and reduced adiponectin levels in the supernatant of treated adipocytes. Moreover, ROS production was significantly elevated after DEHP-exposure. In conclusion, DEHP led to lower grade of adipogenic differentiation in human SGBS-adipocytes under the chosen conditions.