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The activation of genes within the nucleus of eukaryotic cells is a tightly regulated process, orchestrated by a complex interplay of various physical properties and interacting factors. Studying the multitude of components and features that collectively contribute to gene activation has proven challenging due to the complexities of simultaneously visualizing the dynamic and transiently interacting elements that coalesce within the small space occupied by each individual gene. However, various labeling and imaging advances are now starting to overcome this challenge, enabling visualization of gene activation at different lengths and timescales. In this review, we aim to highlight these microscopy-based advances and suggest how they can be combined to provide a comprehensive view of the mechanisms regulating gene activation., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid-Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Confocal microscopy is an invaluable tool for 3D imaging of biological specimens, however, accessibility is often limited to core facilities due to the high cost of the hardware. We describe an inexpensive do-it-yourself (DIY) spinning disk confocal microscope (SDCM) module based on a commercially fabricated chromium photomask that can be added on to a laser-illuminated epifluorescence microscope. The SDCM achieves strong performance across a wide wavelength range (∼400-800 nm) as demonstrated through a series of biological imaging applications that include conventional microscopy (immunofluorescence, small-molecule stains, and fluorescence in situ hybridization) and super-resolution microscopy (single-molecule localization microscopy and expansion microscopy). This low-cost and simple DIY SDCM is well-documented and should help increase accessibility to confocal microscopy for researchers., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During development, progenitors often differentiate many cell generations after receiving signals. These delays must be robust yet tunable for precise population size control. Polycomb repressive mechanisms, involving histone H3 lysine-27 trimethylation (H3K27me3), restrain the expression of lineage-specifying genes in progenitors and may delay their activation and ensuing differentiation. Here, we elucidate an epigenetic switch controlling the T cell commitment gene Bcl11b that holds its locus in a heritable inactive state for multiple cell generations before activation. Integrating experiments and modeling, we identify a mechanism where H3K27me3 levels at Bcl11b, regulated by methyltransferase and demethylase activities, set the time delay at which the locus switches from a compacted, silent state to an extended, active state. This activation delay robustly spans many cell generations, is tunable by chromatin modifiers and transcription factors, and is independent of cell division. With their regulatory flexibility, such timed epigenetic switches may broadly control timing in development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

Experience-dependent changes in the structure of dendritic spines may contribute to learning and memory. Encoded by three genes, the Shank family of postsynaptic scaffold proteins are abundant and enriched in the postsynaptic density (PSD) of central excitatory synapses. When expressed in cultured hippocampal neurons, Shank promotes the maturation and enlargement of dendritic spines. Recently, Shank3 has been genetically implicated in human autism, suggesting an important role for Shank proteins in normal cognitive development. Here, we report the phenotype of Shank1 knock-out mice. Shank1 mutants showed altered PSD protein composition; reduced size of dendritic spines; smaller, thinner PSDs; and weaker basal synaptic transmission. Standard measures of synaptic plasticity were normal. Behaviorally, they had increased anxiety-related behavior and impaired contextual fear memory. Remarkably, Shank1-deficient mice displayed enhanced performance in a spatial learning task; however, their long-term memory retention in this task was impaired. These results affirm the importance of Shank1 for synapse structure and function in vivo, and they highlight a differential role for Shank1 in specific cognitive processes, a feature that may be relevant to human autism spectrum disorders.

Background: The telephone information service of the Cancer Information Service (CIS) historically is most effective in eliciting calls from higher income, white women. This article describes the design and feasibility of a project that tested the use of telephone outcalls to extend the reach of the telephone information service to underserved women., Methods: Neighborhoods throughout Colorado were identified using a geodemographic database (INFORUM) that allowed selection of census block groups according to demographic characteristics. Households were assigned randomly to: (1) a control group; (2) an outcall-only group, which received "cold" telephone outcalls promoting screening mammography; and (3) an advance card plus outcall group, which received a card introducing the program prior to the outcall., Results: The use of INFORUM to target low-income, less educated, and black women was largely successful. While quality of intervention delivery was high, the protocol was labor intensive, requiring an average of 40 min to identify and counsel each eligible woman. The advance card did not increase acceptance of the outcalls., Conclusions: This approach successfully extended the CIS's audience; however, its labor intensity may limit its applicability. Strategies for increasing the efficiency of outcall efforts are suggested., (Copyright 1998 American Health Foundation and Academic Press.)

Background: This study evaluated the impact of a telephone outcall intervention (based on the Transtheoretical Model) on screening mammography behavior among lower income, older women., Methods: A geodemographic database, INFORUM, was used to identify low-income and minority neighborhoods throughout the state of Colorado. Residences were assigned randomly to three study groups: (1) control, (2) outcall only, and (3) advance "invitation" + outcall. Information Specialists of the Cancer Information Service implemented the protocol. Mammography adherence was assessed in telephone interviews conducted 6 months and 2 years after the initial call., Results: Neither intervention had a significant effect on the main outcome, receipt of mammography in the 6-month follow-up period. At 6 months, intentions to have a mammogram were significantly stronger in the intervention groups compared with the control group, particularly among those who were precontemplators at baseline. The 2-year follow-up indicated a small increase in mammography adherence attributable to the advance invitation + outcall, but this effect was restricted to those adherent at baseline. Mammography behavior during the 6-month follow-up period was predicted strongly by decisional balance, intentions, receipt of a physical and clinical breast exam, and previous mammography behavior., Conclusions: The intervention promoted minimal movement in the stages of change for mammography. Outcall interventions may have promise for encouraging repeat mammography behavior, but more intensive interventions are likely to be necessary to promote behavior change among nonadherent women., (Copyright American Health Foundation and Academic Press.)

Over the past 15 years, the Cancer Information Service (CIS) of the National Cancer Institute has generated a substantial body of research. In the present report, this research is reviewed as it pertains to five key questions: 1) Who uses the CIS? 2) What are the topics of inquiry? 3) How do callers learn of the CIS? 4) Do CIS campaigns and unplanned events affect CIS call volume? 5) Can the CIS influence behavior? Major conclusions from this review can be summarized as follows: 1) The vast majority of callers to the CIS are female, White/Anglo, and of middle-class background. 2) Most calls relate directly or indirectly to cancer-treatment issues. 3) A variety of media and sources are reported by CIS callers, with television ranking high across most studies. 4) CIS call volume can be substantially influenced by promotion campaigns and unplanned events. 5) The available evidence suggests that the CIS can influence behavior, especially information-seeking behavior. Recommendations for future research are discussed in light of the results of this review.

This paper identifies the potential applications of information-management technology in the Cancer Information Service national network and describes some innovations of individual network offices in each area. New applications are explored and recommendations are made for coordinating these efforts nationally.