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Type of Publication
3945 Publications
Showing 91-100 of 3945 resultsA theoretical formulation for optically active sum frequency generation (OA-SFG) from isotropic chiral solutions was proposed for molecules with a chiral side chain and an intrinsically achiral chromophore. Adapting an electron correlation model first proposed by Höhn and Weigang for linear optical activity, we presented a dynamic coupling model for OA-SFG near the electronic resonance of the achiral chromophore. As a demonstration, we used this model to explain the observed OA-SFG spectra of a series of amino acids near the electronic resonance of the intrinsically achiral carboxyl group. Our model shows that the nonlinear chiroptical response comes about by the through-space correlative electronic interactions between the chiral side chain and the achiral chromophore, and its magnitude is determined by the position and orientation of the bonds that make up the chiral side chain. Using the bond polarizability values in the literature and the conformations of amino acids obtained from calculation, we were able to reproduce the relative OA-SFG strength from a series of amino acids.
Transcription factor (TF)-directed enhanceosome assembly constitutes a fundamental regulatory mechanism driving spatiotemporal gene expression programs during animal development. Despite decades of study, we know little about the dynamics or order of events animating TF assembly at cis-regulatory elements in living cells and the long-range molecular "dialog" between enhancers and promoters. Here, combining genetic, genomic, and imaging approaches, we characterize a complex long-range enhancer cluster governing Krüppel-like factor 4 (Klf4) expression in naïve pluripotency. Genome editing by CRISPR/Cas9 revealed that OCT4 and SOX2 safeguard an accessible chromatin neighborhood to assist the binding of other TFs/cofactors to the enhancer. Single-molecule live-cell imaging uncovered that two naïve pluripotency TFs, STAT3 and ESRRB, interrogate chromatin in a highly dynamic manner, in which SOX2 promotes ESRRB target search and chromatin-binding dynamics through a direct protein-tethering mechanism. Together, our results support a highly dynamic yet intrinsically ordered enhanceosome assembly to maintain the finely balanced transcription program underlying naïve pluripotency.
Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐ fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐ red chemogenetic reporter of protein‐protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.
Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.
Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2. Whole body cryoslicing, imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an 'Achilles heel' for the early diagnosis of metastatic disease and targeted drug delivery.
Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.
Although different animal species often exhibit extensive variation in many behaviors, typically scientists examine one or a small number of behaviors in any single study. Here, we propose a new framework to simultaneously study the evolution of many behaviors. We measured the behavioral repertoire of individuals from six species of fruit flies using unsupervised techniques and identified all stereotyped movements exhibited by each species. We then fit a Generalized Linear Mixed Model to estimate the intra- and inter-species behavioral covariances, and, by using the known phylogenetic relationships among species, we estimated the (unobserved) behaviors exhibited by ancestral species. We found that much of intra-specific behavioral variation has a similar covariance structure to previously described long-time scale variation in an individual's behavior, suggesting that much of the measured variation between individuals of a single species in our assay reflects differences in the status of neural networks, rather than genetic or developmental differences between individuals. We then propose a method to identify groups of behaviors that appear to have evolved in a correlated manner, illustrating how sets of behaviors, rather than individual behaviors, likely evolved. Our approach provides a new framework for identifying co-evolving behaviors and may provide new opportunities to study the mechanistic basis of behavioral evolution.
Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5’ and 3’ ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.