Effect of pressure on the flow behavior of polybutene

The rheology of submicron thick polymer melt is examined under high normal pressure conditions by a recently developed photobleached‐fluorescence imaging velocimetry technique. In particular, the validity and limitation of Reynold equation solution, which suggests a linear through‐thickness velocity profile, is investigated. Polybutene (PB) is sheared between two surfaces in a point contact. The results presented in this work suggest the existence of a critical pressure below which the through‐thickness velocity profile is close to linear. At higher pressures however, the profile assumes a sigmoidal shape resembling partial plug flow. The departure of the sigmoidal profile from the linear profile increases with pressure, which is indicative of a second‐order phase/glass transition. The nature of the transition is confirmed independently by examining the pressure‐dependent dynamics of PB squeeze films. The critical pressure for flow profile transition varies with molecular weight, which is consistent with the pressure‐induced glass transition of polymer melt

Single-molecule visualization of DNA G-quadruplex formation in live cells.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.Supported by programme grant funding from Cancer Research UK (C9681/A18618, S.B.) core funding from Cancer Research UK (C14303/A17197, S.B.), a Royal Society University Research Fellowship (UF120277 to S.F.L.), Research Professorship (RP150066 to D.K.), a EPSRC (EP/L027631/1 to D.K.) and a BBSRC David Phillips Fellowship (BB/R011605/1 to M.D.A

Genomic and Transcriptional Co-Localization of Protein-Coding and Long Non-Coding RNA Pairs in the Developing Brain

Besides protein-coding mRNAs, eukaryotic transcriptomes include many long non-protein-coding RNAs (ncRNAs) of unknown function that are transcribed away from protein-coding loci. Here, we have identified 659 intergenic long ncRNAs whose genomic sequences individually exhibit evolutionary constraint, a hallmark of functionality. Of this set, those expressed in the brain are more frequently conserved and are significantly enriched with predicted RNA secondary structures. Furthermore, brain-expressed long ncRNAs are preferentially located adjacent to protein-coding genes that are (1) also expressed in the brain and (2) involved in transcriptional regulation or in nervous system development. This led us to the hypothesis that spatiotemporal co-expression of ncRNAs and nearby protein-coding genes represents a general phenomenon, a prediction that was confirmed subsequently by in situ hybridisation in developing and adult mouse brain. We provide the full set of constrained long ncRNAs as an important experimental resource and present, for the first time, substantive and predictive criteria for prioritising long ncRNA and mRNA transcript pairs when investigating their biological functions and contributions to development and disease

Additive manufacturing of solid diffractive optical elements via near index matching

Diffractive optical elements (DOEs) have a wide range of applications in optics and photonics, thanks to their capability to perform complex wavefront shaping in a compact form. However, widespread applicability of DOEs is still limited, because existing fabrication methods are cumbersome and expensive. Here, we present a simple and cost-effective fabrication approach for solid, high-performance DOEs. The method is based on conjugating two nearly refractive index-matched solidifiable transparent materials. The index matching allows for extreme scaling up of the elements in the axial dimension, which enables simple fabrication of a template using commercially available 3D printing at tens-of-micrometer resolution. We demonstrated the approach by fabricating and using DOEs serving as microlens arrays, vortex plates, including for highly sensitive applications such as vector beam generation and super-resolution microscopy using MINSTED, and phase-masks for three-dimensional single-molecule localization microscopy. Beyond the advantage of making DOEs widely accessible by drastically simplifying their production, the method also overcomes difficulties faced by existing methods in fabricating highly complex elements, such as high-order vortex plates, and spectrum-encoding phase masks for microscopy

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.We thank the Royal Society for the University Research Fellowship of S.F.L. (UF120277). This work was kindly funded by the Engineering and Physical Sciences Research Council (EP/M003663/1) and by the Wellcome Trust

Single-molecule imaging of Wnt3A protein diffusion on living cell membranes

Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localisation at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labelled Wnt3A transiently and dynamically associates with the membranes of Drosophila S2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2R+ cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes and represent a new system for investigating the dynamics of Wnt transport.We thank the Royal Society for the University Research Fellowship of S.F.L. (UF120277). We thank the MRC for funding A.A.J’s studentship (MR/J004103/1). We also thank the Bone Research Society, UK for a Barbara Mawer travel award and the Tissue and Cell Engineering Society for Short Scientific Mission award to A.A.J. This work was supported in part by the National Institute of General Medical Sciences, Grant No. R01GM086196 (to W.E.M)

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.This work was funded by The Wellcome Trust, the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and Cancer Research UK. We thank the Wolfson Imaging Centre, University of Oxford, for access to their microscope facility. We would like to thank the Wellcome Trust for the Sir Henry Dale Fellowship of R.A.F. (WT101609MA), the Royal Society for the University Research Fellowship of S.F.L. (UF120277) and acknowledge a GSK Professorship (D.K.). We are also grateful to Doug Tischer (UCSF, US) and Muaz Rushdi (Georgia Tech, US) for their critical comments on the manuscript

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping

We have developed a multi-dimensional super-resolution (md-SR) imaging technique to determine both the localization and the environmental properties of single-molecule fluorescent emitters. The method, termed sPAINT, exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule to enable the mapping of hydrophobicity of biological structures. We first validated the sPAINT method by studying synthetic lipid vesicles of known composition, then applied it to measure the hydrophobicity of amyloid fibrils and oligomers implicated in neurodegenerative diseases, and of the plasma membrane of mammalian cells. sPAINT is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, which enables all the necessary information to be extracted simultaneously from a single image plane. sPAINT enables the hydrophobicity of surfaces to be mapped at the nanoscale in a dynamic fashion.Medical Research Council (Grant ID: MR/K015850/1), Engineering and Physical Sciences Research Council, Royal Society (University Research Fellowship, Grant ID: UF120277), Augustus Newman Foundation, Cambridge Advanced Imaging Centre, Christ’s Colleg

lincRNAs act in the circuitry controlling pluripotency and differentiation

Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.Broad InstituteHarvard UniversityNational Human Genome Research Institute (U.S.)Merkin Family Foundation for Stem Cell Researc

A Large Fraction of Extragenic RNA Pol II Transcription Sites Overlap Enhancers

A substantial fraction of extragenic Pol II transcription sites coincides with transcriptional enhancers, which may be relevant for functional annotation of mammalian genomes