ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Learning Continuous 2D Diffusion Maps from Particle Trajectories without Data Binning.

Diffusion coefficients often vary across regions, such as cellular membranes, and quantifying their variation can provide valuable insight into local membrane properties such as composition and stiffness. Toward quantifying diffusion coefficient spatial maps and uncertainties from particle tracks, we use a Bayesian method and place Gaussian Process (GP) Priors on the maps. For the sake of computational efficiency, we leverage inducing point methods on GPs arising from the mathematical structure of the data giving rise to non-conjugate likelihood-prior pairs. We analyze both synthetic data, where ground truth is known, as well as data drawn from live-cell single-molecule imaging of membrane proteins. The resulting tool provides an unsupervised method to rigorously map diffusion coefficients continuously across membranes without data binning.

Ileoanal pouch revision and excision surgery in a newly established pouch center: requirements and costs for service provision.

Complications of ileoanal pouch surgery affecting function and quality of life may require surgical correction or pouch excision. The management of patients with pouch dysfunction requires a multidisciplinary approach and demand for service provision include multiple healthcare professionals and resources. The aim of this study is to present the service requirements, and surgical outcomes for redo pouch surgery and pouch excision, with cost analysis of the required resources. All patients undergoing surgery for revision or excision of the ileoanal pouch from June 2021 to May 2023 were prospectively included. Patient undergoing only diagnostic procedures, or perineal procedures were excluded. Outcomes within 30 days of surgery were collected, including readmissions and re-operations. Cost analysis of all investigations, outpatient appointments and procedures prior to pouch revision or pouch excision was conducted. Twenty patients were included during the 24 months study period: 13 underwent abdominal revisional pouch surgery, 7 had ileoanal pouch excision. 15 patients (75%) were tertiary referrals from other hospitals in the UK. The median interval between index IPAA surgery and revision was 113 months. Three multidisciplinary clinical appointments, two imaging modalities, and at least one invasive day-surgery procedure were required for each patient prior to surgery. Expertise and infrastructure are needed for indication and peri-operative management of patients with pouch dysfunction requiring pouch revision or pouch excision. We estimated a starting cost of £22.605 ($29.589) for provision of pouch revision or excision surgery for investigations and treatments from referral to the pouch unit to surgery. This likely represents an underestimate as only accounts for procedures performed since referral with pouch dysfunction.

Patient-reported Outcome Measures in Ileoanal Pouch Surgery: a Systematic Review.

OBJECTIVE: To summarise frequency, type, and heterogeneity of patient-reported outcomes measures [PROMs] in papers reporting on outcomes after ileal pouch anal anastomosis [IPAA]. BACKGROUND: Prevalence of ulcerative colitis [UC] has risen in Western countries, and one in three patients requires surgery. IPAA is a frequently performed procedure for UC, and a lack of standardisation is manifest in reporting outcomes for inflammatory bowel disease [IBD] despite the clear need for inclusion of PROMs as primary outcomes in IBD trials. METHODS: Scopus, Pubmed, and Web of Science databases were searched from January 2010 to January 2023 for studies investigating outcomes in IPAA surgery. The primary outcome was the proportion of studies reporting outcomes for IPAA surgery for UC, which included PROMs. RESULTS: The search identified a total of 8028 studies which, after de-duplication and exclusion, were reduced to 79 articles assessing outcomes after IPAA surgery. In all 44 [55.7%] reported PROMs, with 23 including validated questionnaires and 21 papers using authors' questions, 22 different PROMs were identified, with bowel function as the most investigated item. The majority of studies [67/79, 85%] were retrospective, only 14/79 [18%] were prospective papers and only two were [2.5%] randomised, controlled trials. CONCLUSIONS: Only half of the papers reviewed used PROMs. The main reported item is bowel function and urogenital, social, and psychological functions are the most neglected. There is lack of standardisation for use of PROMs in IPAA. Complexity of UC and of outcomes after IPAA demands a change in clinical practice and follow-up, given how crucial PROMs are, compared with their non-routine use.

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.

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.

Patient-reported outcome measures and surgery for Crohn's disease: systematic review.

BACKGROUND/AIMS: Crohn's disease is an inflammatory bowel disease with up to 50 per cent of patients requiring surgery within 10 years of diagnosis. Patient-reported outcome measures (PROMs) are vital to monitor and assess patient health-related quality of life (HRQoL). This systematic review aims to evaluate PROMs within studies for perioperative Crohn's disease patients. METHODS: Articles from MEDLINE, Embase, Emcare and CINAHL databases were searched to find studies relating to the assessment of HRQoL in perioperative Crohn's disease patients using PROMs and patient-reported experience measures (PREMs) from 1st January 2015 to 22nd October 2023. Bias was assessed using the ROBINS-I tool was used for non-randomized interventional studies and the Cochrane RoB2 tool was used for randomized trials. RESULTS: 1714 journal articles were filtered down to eight studies. Six studies focused on ileocaecal resection, one on perianal fistulas and one on the effects of cholecystectomy on patients with Crohn's disease. Within these articles, ten different PROM tools were identified (8 measures of HRQoL and 2 measures of functional outcome). Overall improvements in patient HRQoL pre- to postoperative for ileocaecal Crohn's disease were found in both paediatric and adult patients. Outcomes were comparable in patients in remission, with or without stoma, but were worse in patients with a stoma and active disease. CONCLUSION: There are significant variations in how PROMs are used to evaluate perioperative Crohn's disease outcomes and a need for consensus on how tools are used. Routine assessments using an internationally accepted online platform can be used to monitor patients and support areas of treatment pathways that require further support to ensure high standards of care. They also enable future statistical comparisons in quantitative reviews and meta-analyses.

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.

Practice of proctology among general surgery residents and young specialists in Italy: a snapshot survey.

Anal diseases are very common and, in most of the cases, require surgery of minor or medium complexity, and, therefore, are among the most accessible diseases for surgeons in training. Aim of this study is to investigate the status of the training in proctology in Italy. A 31-items questionnaire was administered to residents and young specialists (≤ 2 years) in general surgery, using mailing lists, and the social media accounts of the Italian Society of Colorectal Surgery. Answers from 338 respondents (53.8% males) were included in the final analysis. Overall, 252 respondents (74.5%) were residents and 86 (25.5%) young specialists. Two hundred and fifty-five (75.4%) respondents practiced proctology for the first time early on during their postgraduate training, but only 19.5% did it continuously for ≥ 24 months. Almost all respondents (334; 98.8%) had the chance to participate in proctological procedures, 205 (60.5%) as first surgeon. This percentage decreases according to the complexity of the surgery. In fact, only 11 (3.3%) and 24 (7.1%) of the respondents were allowed to be the first surgeon in more complex proctological diseases such as surgery for rectal prolapse and fecal incontinence. The present survey suggests that, in Italy, most surgeons in training deal with anal diseases. However, only few of them could achieve sufficient professional skills in the management of proctological diseases to be able to practice autonomously as young specialists.

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.

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.

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

Roadmap on Deep Learning for Microscopy.

Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.

Repeated surgery for recurrent Crohn's disease: does the outcome keep worsening operation after operation? A comparative study of 1224 consecutive procedures.

Complicated Crohn's disease (CD) will require surgical treatment during patients' lifetime, with a considerable recurrence rate requiring additional surgery. The present study is a retrospective analysis of a prospectively maintained database in an IBD Tertiary Centre that included all the consecutive, unselected patients undergoing surgery for CD between 1993 and 2019. Patients treated with small bowel resections, colonic resections, conventional and non-conventional strictureplasties were considered. The aim was to evaluate morbidity and long-term recurrence of repeated surgery. Among the population included, the following procedures were performed: 713 (58.2%) primary surgery (group S1), 325 (26.5%) first recurrence (group S2), and 186 (15.3%) multiple recurrences (group S3). Patients undergoing repeat surgery were older (p < 0.0001) and had a longer disease duration (p < 0.0001), extended disease (p = 0.0001), shorter time frame to first surgery (p < 0.0001), nutritional impairment (p < 0.0001), and a history of aggressive medical therapy (p = 0.04). Patients undergoing surgery for recurrences required higher complexity level surgery, with more conservative approaches (p = 0.0004) and a higher ostomy number (p = 0.06). Recurrent patients had higher short bowel syndrome rate (p < 0.0001), higher minor (p = 0.04) but not major (p = 0.2) postoperative complications rate. The 10-year surgical recurrence rate was 18% for group S1, 27% for S2, and 48% for S3, with significant differences at the log-rank test. Repeated surgery for complicated CD was associated with an increased rate of minor, but not major complications, requiring high-risk surgery, with a major ostomy rate and short bowel syndrome, and is associated with an increased long-term surgical recurrence, even on strictureplasty sites.

Objective comparison of methods to decode anomalous diffusion.

Deviations from Brownian motion leading to anomalous diffusion are found in transport dynamics from quantum physics to life sciences. The characterization of anomalous diffusion from the measurement of an individual trajectory is a challenging task, which traditionally relies on calculating the trajectory mean squared displacement. However, this approach breaks down for cases of practical interest, e.g., short or noisy trajectories, heterogeneous behaviour, or non-ergodic processes. Recently, several new approaches have been proposed, mostly building on the ongoing machine-learning revolution. To perform an objective comparison of methods, we gathered the community and organized an open competition, the Anomalous Diffusion challenge (AnDi). Participating teams applied their algorithms to a commonly-defined dataset including diverse conditions. Although no single method performed best across all scenarios, machine-learning-based approaches achieved superior performance for all tasks. The discussion of the challenge results provides practical advice for users and a benchmark for developers.

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.

Falciform Ligament Flap as Crural Buttress in Laparoscopic Hiatal Hernia Repair.

Background: Crural repair is an essential technical component in laparoscopic hiatal hernia surgery, but there is no consensus regarding the optimal method to prevent postoperative hernia recurrence. Mesh augmentation, especially with permanent materials, is associated with dysphagia and complications. The rotational falciform ligament flap (FLF) has been reported to be effective in reinforcing standard suture closure of the hiatus. Materials and Methods: Patients with primary or secondary hiatal hernia in whom FLF was used to buttress the hiatus repair were included. The FLF was dissected from the anterior abdominal wall, detached from the umbilical area, and transposed below the left lateral liver segment to buttress the cruroplasty. Indocyanine green fluorescence was used to assess vascularization of the flap before and after mobilization. Results: Eighteen consecutive patients underwent laparoscopic FLF cruroplasty reinforcement between October 2019 and January 2021. Indications were primary hiatal hernia (n = 9), recurrent hiatal hernia (n = 4), postsleeve gastrectomy hernia (n = 1), prophylactic hiatal repair during esophagectomy and gastric conduit reconstruction (n = 2), and postesophagectomy hernia (n = 2). All flaps were well vascularized and covered the entire hiatal area. There was no morbidity. At a median follow-up of 8 months (range 3-15), the symptomatic and quality of life scores significantly improved compared with baseline (P < .001), and no anatomic hernia recurrences were detected. Conclusions: FLF is safe for crural buttress and is a viable alternative to mesh in laparoscopic hiatal hernia surgery.

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.