Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo.

Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state.

Separating Actin-Dependent Chemokine Receptor Nanoclustering from Dimerization Indicates a Role for Clustering in CXCR4 Signaling and Function.

A current challenge in cell motility studies is to understand the molecular and physical mechanisms that govern chemokine receptor nanoscale organization at the cell membrane, and their influence on cell response. Using single-particle tracking and super-resolution microscopy, we found that the chemokine receptor CXCR4 forms basal nanoclusters in resting T cells, whose extent, dynamics, and signaling strength are modulated by the orchestrated action of the actin cytoskeleton, the co-receptor CD4, and its ligand CXCL12. We identified three CXCR4 structural residues that are crucial for nanoclustering and generated an oligomerization-defective mutant that dimerized but did not form nanoclusters in response to CXCL12, which severely impaired signaling. Overall, our data provide new insights to the field of chemokine biology by showing that receptor dimerization in the absence of nanoclustering is unable to fully support CXCL12-mediated responses, including signaling and cell function in vivo.

Inferring pointwise diffusion properties of single trajectories with deep learning.

To characterize the mechanisms governing the diffusion of particles in biological scenarios, it is essential to accurately determine their diffusive properties. To do so, we propose a machine-learning method to characterize diffusion processes with time-dependent properties at the experimental time resolution. Our approach operates at the single-trajectory level predicting the properties of interest, such as the diffusion coefficient or the anomalous diffusion exponent, at every time step of the trajectory. In this way, changes in the diffusive properties occurring along the trajectory emerge naturally in the prediction and thus allow the characterization without any prior knowledge or assumption about the system. We first benchmark the method on synthetic trajectories simulated under several conditions. We show that our approach can successfully characterize both abrupt and continuous changes in the diffusion coefficient or the anomalous diffusion exponent. Finally, we leverage the method to analyze experiments of single-molecule diffusion of two membrane proteins in living cells: the pathogen-recognition receptor DC-SIGN and the integrin α5ß1. The analysis allows us to characterize physical parameters and diffusive states with unprecedented accuracy, shedding new light on the underlying mechanisms.

Broadband Plasmonic Nanoantennas for Multi-Color Nanoscale Dynamics in Living Cells.

Recently, the implementation of plasmonic nanoantennas has opened new possibilities to investigate the nanoscale dynamics of individual biomolecules in living cells. However, studies so far have been restricted to single molecular species as the narrow wavelength resonance of gold-based nanostructures precludes the simultaneous interrogation of different fluorescently labeled molecules. Here, broadband aluminum-based nanoantennas carved at the apex of near-field probes are exploited to resolve nanoscale-dynamic molecular interactions on living cell membranes. Through multicolor excitation, the authors simultaneously recorded fluorescence fluctuations of dual-color labeled transmembrane receptors known to form nanoclusters. Fluorescence cross-correlation studies revealed transient interactions between individual receptors in regions of ≈60 nm. Moreover, the high signal-to-background ratio provided by the antenna illumination allowed the authors to directly detect fluorescent bursts arising from the passage of individual receptors underneath the antenna. Remarkably, by reducing the illumination volume below the characteristic receptor nanocluster sizes, the molecular diffusion within nanoclusters is resolved and distinguished from nanocluster diffusion. Spatiotemporal characterization of transient interactions between molecules is crucial to understand how they communicate with each other to regulate cell function. This work demonstrates the potential of broadband photonic antennas to study multi-molecular events and interactions in living cell membranes with unprecedented spatiotemporal resolution.

Ten steps for ileoanal pouch anastomosis.

AIM: Appropriate patient selection, surgical technique, and follow-up pathways can provide optimal functional outcomes and good quality of life in many patients undergoing ileoanal pouch surgery. The aim of this study was to demonstrate the standardised approach to ileoanal pouch formation that we have developed in our pouch surgery centre. METHODS: We developed a structured approach to laparoscopic proctectomy with ileoanal pouch anastomosis formation, divided into 10 different steps. All patients referred to our centre from January 2020 to December 2022 for ulcerative colitis were included in the study. RESULTS: A total of 38 consecutive patients underwent ileal pouch-anal anastomosis (IPAA) surgery. All procedures were completed laparoscopically with one conversion to open (2.6%). A total of 13 patients had postoperative complications within 30 days of surgery (34.2%), with six (15.8%) being Clavien Dindo class 3 or higher. Median follow-up length was 18 months (range 2-30). Median number of bowel movements in 24 h at 12 months post-surgery was 4 (range 1-11). CONCLUSIONS: Our modular 10 steps approach could provide a standardised framework to surgeons in the learning curve. IPAA is a complex surgical procedure with significant postoperative morbidity. Our stepwise approach resulted in a high rate of minimally invasive surgery and could facilitate introduction of the technique.

Stapled disc excision for the repair of intraoperative rectal injury during robotic surgery for deep infiltrating endometriosis.

INTRODUCTION: Intraoperative rectal perforation is an uncommon complication of pelvic surgery, which can be life-threatening and often leads to high morbidity and stoma formation rate. PURPOSE: No consensus has been reached regarding a standard of care for intraoperative iatrogenic pelvic injury. This article presents a technique for a stapled repair to completely resect a full-thickness low rectal perforation during robotic surgery for advanced endometriosis and avoid a high-risk colorectal anastomosis and the possible need for stoma formation. CONCLUSION: Stapled discoid excision is a novel and safe technique for the repair of intraoperative rectal injuries, showing multiple benefits compared to the standard colorectal resection with or without anastomosis.

Assessment of the ileoanal pouch for the colorectal surgeon.

INTRODUCTION: Many pouch complications following ileoanal pouch surgery have an inflammatory or mechanical nature, and specialist colorectal surgeons are required to assess the anatomy of the ileoanal pouch in multiple settings. In this study, we report our stepwise clinical and endoscopic assessment of the patient with an ileoanal pouch. METHODS: The most common configuration of the ileoanal pouch is a J-pouch, and the stapled anastomosis is more frequently performed than a handsewn post-mucosectomy. A structured clinical and endoscopic assessment of the ileoanal pouch must provide information on 7 critical areas: anus and perineum, rectal cuff, pouch anal anastomosis, pouch body, blind end of the pouch, pouch inlet and pre-pouch ileum. RESULTS: We have developed a structured pro forma for step-wise assessment of the ileoanal pouch, according to 7 essential areas to be evaluated, biopsied and reported. The structured assessment of the ileoanal pouch in 102 patients allowed reporting of abnormal findings in 63 (61.7%). Strictures were diagnosed in 27 patients (26.4%), 3 pouch inlet strictures, 21 pouch anal anastomosis strictures, and 3 pre-pouch ileum strictures. Chronic, recurrent pouchitis was diagnosed in 9 patients, whilst 1 patient had Crohn's disease of the pouch. CONCLUSIONS: Detailed clinical history, assessment of symptoms and multidisciplinary input are all essential for the care of patients with an ileoanal pouch. We present a comprehensive reporting pro forma for initial clinical assessment of the patient with an ileoanal pouch, with the aim to guide further investigations and inform multidisciplinary decision-making.

Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration.

The leukocyte-specific ß2-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.

A toolkit for the quantitative evaluation of chronic wounds evolution for early detection of non-healing wounds.

BACKGROUND: Chronic wounds resulting from a number of conditions do not heal properly and can pose serious health problems. Beyond clinician visual inspection, an objective evaluation of the wound is required to assess wound evolution and the effectiveness of therapies. AIM: Our objective is to provide a methodology for the analysis of wound area vs. time for the early prediction of non-healing wounds evolution. METHODS: We propose a two-step approach consisting of: i) wound area quantification from planimetries and ii) classification of wound healing through the inference of characteristic parameters. For the first step, we describe a user-friendly software (Woundaries) to automatically calculate the wound area and other geometric parameters from hand-traced planimetries. For the second, we use a procedure for the objective classification of wound time evolution and the early assessment of treatment efficacy. The methodology was tested on simulations and retrospectively applied to data from 85 patients to compare the effect of a biological therapy with respect to general basic therapeutics. RESULTS: Woundaries provides measurements of wound surface equivalent to a validated device. The two-step methodology allows to determine if a wound is healing with high sensitivity, even with limited amount of data. Therefore, it allows the early assessment of the efficacy of a therapy. CONCLUSION: The performance of this methodology for the quantification and the objective evaluation of wound area evolution suggest it as a useful toolkit to assist clinicians in the early assessment of the efficacy of treatments, leading to a timely change of therapy.

Direct and Fast Assessment of Antimicrobial Surface Activity Using Molecular Dynamics Simulation and Time-Lapse Imaging.

With the alarming rise of antimicrobial resistance, studies on bacteria-surface interactions are both relevant and timely. Scanning electron microscopy and colony forming unit counting are commonly used techniques but require sophisticated sample preparation and long incubation time. Here, we present a direct method based on molecular dynamics simulation of nanostructured surfaces providing in silico predictions, complemented with time-lapse fluorescence imaging to study live interactions of bacteria at the membrane-substrate level. We evaluate its effectiveness in predicting and statistically analyzing the temporal evolution and spatial distribution of prototypical bacteria with costained nucleoids and membranes (E. coli) on surfaces with nanopillars. We observed cell reorientation, clustering, membrane damage, growth inhibition, and in the extreme case of hydrocarbon-coated nanopillars, this was followed by cell disappearance, validating the obtained simulation results. Contrary to commonly used experimental methods, microscopy data are fast processed, in less than 1 h. In particular, the bactericidal effects can be straightforwardly detected and correlated with surface morphology and/or wettability.

A DNA origami platform for quantifying protein copy number in super-resolution.

Single-molecule-based super-resolution microscopy offers researchers a unique opportunity to quantify protein copy number with nanoscale resolution. However, while fluorescent proteins have been characterized for quantitative imaging using calibration standards, similar calibration tools for immunofluorescence with small organic fluorophores are lacking. Here we show that DNA origami, in combination with GFP antibodies, is a versatile platform for calibrating fluorophore and antibody labeling efficiency to quantify protein copy number in cellular contexts using super-resolution microscopy.

Bayesian analysis of data from segmented super-resolution images for quantifying protein clustering.

Super-resolution imaging techniques have largely improved our capabilities to visualize nanometric structures in biological systems. Their application further permits the quantitation relevant parameters to determine the molecular organization and stoichiometry in cells. However, the inherently stochastic nature of fluorescence emission and labeling strategies imposes the use of dedicated methods to accurately estimate these parameters. Here, we describe a Bayesian approach to precisely quantitate the relative abundance of molecular aggregates of different stoichiometry from segmented images. The distribution of proxies for the number of molecules in a cluster, such as the number of localizations or the fluorescence intensity, is fitted via a nested sampling algorithm to compare mixture models of increasing complexity and thus determine the optimum number of mixture components and their weights. We test the performance of the algorithm on in silico data as a function of the number of data points, threshold, and distribution shape. We compare these results to those obtained with other statistical methods, showing the improved performance of our approach. Our method provides a robust tool for model selection in fitting data extracted from fluorescence imaging, thus improving the precision of parameter determination. Importantly, the largest benefit of this method occurs for small-statistics or incomplete datasets, enabling an accurate analysis at the single image level. We further present the results of its application to experimental data obtained from the super-resolution imaging of dynein in HeLa cells, confirming the presence of a mixed population of cytoplasmic single motors and higher-order structures.

Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration.

The use of super-resolution microscopy in recent years has revealed that proteins often form small assemblies inside cells and are organized in nanoclusters. However, determining the copy number of proteins within these nanoclusters constitutes a major challenge because of unknown labeling stoichiometries and complex fluorophore photophysics. We previously developed a DNA-origami-based calibration approach to extract protein copy number from super-resolution images. However, the applicability of this approach is limited by the fact that the calibration is dependent on the specific labeling and imaging conditions used in each experiment. Hence, the calibration must be repeated for each experimental condition, which is a formidable task. Here, using cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample, we demonstrate that the DNA-origami-based calibration data we previously generated can be extended to super-resolution images taken under different experimental conditions, enabling the quantification of any green-fluorescent-protein-fused protein of interest. To do so, we first quantified the copy number of dynein motors within nanoclusters in the cytosol and along the microtubules. Interestingly, this quantification showed that dynein motors form assemblies consisting of more than one motor, especially along microtubules. This quantification enabled us to use the HeLa IC74 cells as a reference sample to calibrate and quantify protein copy number independently of labeling and imaging conditions, dramatically improving the versatility and applicability of our approach.

Inhomogeneous membrane receptor diffusion explained by a fractional heteroscedastic time series model.

Single particle tracking experiments have recently uncovered that the motion of cell membrane components can undergo changes of diffusivity as a result of the heterogeneous environment, producing subdiffusion and nonergodic behavior. In this paper, we show that an autoregressive fractionally integrated moving average (ARFIMA) with noise given by generalized autoregressive conditional heteroscedasticity (GARCH) can describe inhomogeneous diffusion in the cell membrane, where the ARFIMA process models anomalous diffusion and the GARCH process explains a fluctuating diffusion parameter.

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells.

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such "tonic" activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.

PLANT: A Method for Detecting Changes of Slope in Noisy Trajectories.

Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells.

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 µs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Lateral Mobility and Nanoscale Spatial Arrangement of Chemokine-activated α4ß1 Integrins on T Cells.

Chemokine stimulation of integrin α4ß1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4ß1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and superresolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4ß1integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4ß1activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-ß1antibody and by increased talin-ß1 association. CXCL12-dependent α4ß1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a 5-fold increase in immobile integrins compared with unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4ß1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Superresolution imaging showed that the nanoscale organization of high-affinity α4ß1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4ß1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4ß1 integrins regulates T lymphocyte adhesion.