Most human transcription factor (TF) genes encode multiple protein isoforms differing in DNA-binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators," both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

Cancer development and progression are generally associated with gene dysregulation, often resulting from changes in the transcription factor (TF) sequence or expression. Identifying key TFs involved in cancer gene regulation provides a framework for potential new therapeutics. This study presents a large-scale cancer gene TF-DNA interaction network, as well as an extensive promoter clone resource for future studies. Highly connected TFs bind to promoters of genes associated with either good or poor cancer prognosis, suggesting that strategies aimed at shifting gene expression balance between these two prognostic groups may be inherently complex. However, we identified potential for oncogene-targeted therapeutics, with half of the tested oncogenes being potentially repressed by influencing specific activators or bifunctional TFs. Finally, we investigate the role of intrinsically disordered regions within the key cancer-related TF ESR1 in DNA binding and transcriptional activity, and found that these regions can have complex trade-offs in TF function. Altogether, our study broadens our knowledge of the TFs involved in cancer gene regulation and provides a valuable resource for future studies and therapeutics.

Cooperativity and antagonism between transcription factors (TFs) can drastically modify their binding to regulatory DNA elements. While mapping these relationships between TFs is important for understanding their context-specific functions, existing approaches either rely on DNA binding motif predictions, interrogate one TF at a time, or study individual TFs in parallel. Here, we introduce paired yeast one-hybrid (pY1H) assays to detect cooperativity and antagonism across hundreds of TF-pairs at DNA regions of interest. We provide evidence that a wide variety of TFs are subject to modulation by other TFs in a DNA region-specific manner. We also demonstrate that TF-TF relationships are often affected by alternative isoform usage and identify cooperativity and antagonism between human TFs and viral proteins from human papillomaviruses, Epstein-Barr virus, and other viruses. Altogether, pY1H assays provide a broadly applicable framework to study how different functional relationships affect protein occupancy at regulatory DNA regions.

Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have characterized and benchmarked a novel detection technique for scanning electron microscopy: optical scanning transmission electron microscopy (OSTEM). A qualitative and quantitative comparison was performed between OSTEM, secondary and backscattered electron detection and annular dark field detection in scanning transmission electron microscopy. Our analysis shows that OSTEM produces images similar to backscattered electron detection in terms of contrast, resolution and signal-to-noise ratio. OSTEM can complement large scale imaging with (scanning) transmission electron microscopy and has the potential to speed up imaging in single-beam scanning electron microscope.

Journal Article Characterization and Optimization of OSTEM; A Novel Detection Method for Single- and Multi-Beam Scanning Electron Microscopy Get access Arent Kievits, Arent Kievits Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Corresponding author: A.J.Kievits@tudelft.nl Search for other works by this author on: Oxford Academic Google Scholar Job Fermie, Job Fermie Delmic B.V. - Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Peter Duinkerken, Peter Duinkerken Department of Cell Biology, University Medical Centre Groningen- Groningen, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ryan Lane, Ryan Lane Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Elizabeth Carroll, Elizabeth Carroll Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ben Giepmans, Ben Giepmans Department of Cell Biology, University Medical Centre Groningen- Groningen, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jacob Hoogenboom Jacob Hoogenboom Department of Imaging Physics, Delft University of Technology – Delft, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1458–1460, https://doi.org/10.1017/S143192762200592X Published: 01 August 2022

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server-based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.

Journal Article Organelle Segmentation Facilitated by Correlative Light Microscopy Data Get access Ryan Lane, Ryan Lane Delft University of Technology, Department of Imaging Physics, Delft, The Netherlands Corresponding author: r.i.lane@tudelft.nl Search for other works by this author on: Oxford Academic Google Scholar Luuk Balkenende, Luuk Balkenende Delft University of Technology, Department of Imaging Physics, Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Simon van Staalduine, Simon van Staalduine Delft University of Technology, Department of Imaging Physics, Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Anouk H G Wolters, Anouk H G Wolters University Medical Center Groningen, Department of Biomedical Sciences of Cells and Systems, Groningen, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Ben N G Giepmans, Ben N G Giepmans University Medical Center Groningen, Department of Biomedical Sciences of Cells and Systems, Groningen, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Lennard Voortman, Lennard Voortman Leiden University Medical Center, Department of Cell and Chemical Biology, Leiden, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jacob Hoogenboom Jacob Hoogenboom Delft University of Technology, Department of Imaging Physics, Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1518–1520, https://doi.org/10.1017/S1431927622006110 Published: 01 August 2022

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.

The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.

Volume electron microscopy (EM) has revolutionized the way in which biologists understand intraand intercellular systems. Due to the lack of biological specificity, however, interpretation of the data often requires tedious expert analysis and annotation. For this reason fluorescence microscopy (FM) is often used in conjunction with EM, complementing structural data with targeted biological labels. The downside, however is that sample preparation protocols are often laborious, time-consuming, and potentially damaging to the sample. Correlating these multi-modal datasets presents another challenge, which is compounded when registering across large spatial extents and then again for 3D.

Volume electron microscopy (EM) has progressively become a driving force in exploring and analysing three-dimensional biological structures across ever-increasing spatial scales.While advances in technology and automation have revolutionized imaging capabilities, low throughput persists as the primary limitation to achieving larger and larger volumes [1].One strategy for increasing throughput is to combine a fluorescence and electron microscope together into one integrated system.Doing so provides the advantage of being able to use fluorescence expression as a guide for selecting regions of interest for high resolution EM imaging [2].

integrated microscope.Given the scalability of our method, we believe our procedure can pave the way for future large-scale correlative acquisitions-offering a promising approach for the reconstruction of complex biological systems.

We present photometric data of the classical nova, V723 Cas (Nova Cas 1995), over a span of 10 years (2006 through 2016) taken with the 0.9 m telescope at Lowell Observatory, operated as the National Undergraduate Research Observatory (NURO) on Anderson Mesa near Flagstaff, Arizona. A photometric analysis of the data produced light curves in the optical bands (Bessel B, V, and R filters). The data analyzed here reveal an asymmetric light curve (steep rise to maximum, followed by a slow decline to minimum), the overall structure of which exhibits pronounced evolution including a decrease in magnitude from year to year, at the rate of ∼0.15 mag yr−1. We model these data with an irradiated secondary and an accretion disk with a hot spot using the eclipsing binary modeling program Nightfall. We find that we can model reasonably well each season of observation by changing very few parameters. The longitude of the hot spot on the disk and the brightness of the irradiated spot on the companion are largely responsible for the majority of the observed changes in the light curve shape and amplitude until 2009. After that, a decrease in the temperature of the white dwarf is required to model the observed light curves. This is supported by Swift/X-Ray Telescope observations, which indicate that nuclear fusion has ceased, and that V723 Cas is no longer detectable in the X-ray.