A genetic and microscopy toolkit for manipulating and monitoring regeneration in Macrostomum lignano.

Live imaging of regenerative processes can reveal how animals restore their bodies after injury through a cascade of dynamic cellular events. Here, we present a comprehensive toolkit for live imaging of tissue regeneration in the flatworm Macrostomum lignano, including a high-throughput cloning pipeline, targeted cellular ablation, and advanced microscopy solutions. Using tissue-specific reporter expression, we examine how various structures regenerate. Enabled by a custom luminescence/fluorescence microscope, we overcome intense stress-induced autofluorescence to demonstrate genetic cellular ablation and reveal the limited regenerative capacity of neurons and their essential role during wound healing, contrasting muscle cells' rapid regeneration after ablation. Finally, we build an open-source tracking microscope to continuously image freely moving animals throughout the week-long process of regeneration, quantifying kinetics of wound healing, nerve cord repair, body regeneration, growth, and behavioral recovery. Our findings suggest that nerve cord reconnection is highly robust and proceeds independently of regeneration.

Flexible use of conserved motif vocabularies constrains genome access in cell type evolution.

Cell types evolve into a hierarchy with related types grouped into families. How cell type diversification is constrained by the stable separation between families over vast evolutionary times remains unknown. Here, integrating single-nucleus multiomic sequencing and deep learning, we show that hundreds of sequence features (motifs) divide into distinct sets associated with accessible genomes of specific cell type families. This division is conserved across highly divergent, early-branching animals including flatworms and cnidarians. While specific interactions between motifs delineate cell type relationships within families, surprisingly, these interactions are not conserved between species. Consistently, while deep learning models trained on one species can predict accessibility of other species' sequences, their predictions frequently rely on distinct, but synonymous, motif combinations. We propose that long-term stability of cell type families is maintained through genome access specified by conserved motif sets, or 'vocabularies', whereas cell types diversify through flexible use of motifs within each set.

Adaptive robustness through incoherent signaling mechanisms in a regenerative brain.

Animal behavior emerges from collective dynamics of neurons, making it vulnerable to damage. Paradoxically, many organisms exhibit a remarkable ability to maintain significant behavior even after large-scale neural injury. Molecular underpinnings of this extreme robustness remain largely unknown. Here, we develop a quantitative pipeline to measure long-lasting latent states in planarian flatworm behaviors during whole-brain regeneration. By combining >20,000 animal trials with neural network modeling, we show that long-range volumetric peptidergic signals allow the planarian to rapidly restore coarse behavior output after large perturbations to the nervous system, while slow restoration of small-molecule neuromodulator functions refines precision. This relies on the different time and length scales of neuropeptide and small-molecule transmission to generate incoherent patterns of neural activity that competitively regulate behavior. Controlling behavior through opposing communication mechanisms creates a more robust system than either alone and may serve as a generalizable approach for constructing robust neural networks.

Regeneration in the absence of canonical neoblasts in an early branching flatworm.

The remarkable regenerative abilities of flatworms are closely linked to neoblasts - adult pluripotent stem cells that are the only division-competent cell type outside of the reproductive system. Although the presence of neoblast-like cells and whole-body regeneration in other animals has led to the idea that these features may represent the ancestral metazoan state, the evolutionary origin of both remains unclear. Here we show that the catenulid Stenostomum brevipharyngium, a member of the earliest-branching flatworm lineage, lacks conventional neoblasts despite being capable of whole-body regeneration and asexual reproduction. Using a combination of single-nuclei transcriptomics, in situ gene expression analysis, and functional experiments, we find that cell divisions are not restricted to a single cell type and are associated with multiple fully differentiated somatic tissues. Furthermore, the cohort of germline multipotency genes, which are considered canonical neoblast markers, are not expressed in dividing cells, but in the germline instead, and we experimentally show that they are neither necessary for proliferation nor regeneration. Overall, our results challenge the notion that canonical neoblasts are necessary for flatworm regeneration and open up the possibility that neoblast-like cells may have evolved convergently in different animals, independent of their regenerative capacity.

Ultrafast distant wound response is essential for whole-body regeneration.

Injury induces systemic responses, but their functions remain elusive. Mechanisms that can rapidly synchronize wound responses through long distances are also mostly unknown. Using planarian flatworms capable of whole-body regeneration, we report that injury induces extracellular signal-regulated kinase (Erk) activity waves to travel at a speed 10-100 times faster than those in other multicellular tissues. This ultrafast propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. The morphological properties of muscles allow them to act as superhighways for propagating and disseminating wound signals. Inhibiting Erk propagation prevents tissues distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues shortly after the first injury. Our findings provide a mechanism for long-range signal propagation in large, complex tissues to coordinate responses across cell types and highlight the function of feedback between spatially separated tissues during whole-body regeneration.

Expansion spatial transcriptomics.

Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the density of the array. Here we present expansion spatial transcriptomics to overcome this limitation by clearing and expanding tissue prior to capturing the entire polyadenylated transcriptome with an enhanced protocol. This approach enables us to achieve higher spatial resolution while retaining high library quality, which we demonstrate using mouse brain samples.

Ultrafast and long-range coordination of wound responses is essential for whole-body regeneration.

Injury induces systemic, global responses whose functions remain elusive. In addition, mechanisms that rapidly synchronize wound responses through long distances across the organismal scale are mostly unknown. Using planarians, which have extreme regenerative ability, we report that injury induces Erk activity to travel in a wave-like manner at an unexpected speed (∻1 mm/h), 10-100 times faster than those measured in other multicellular tissues. This ultrafast signal propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. Combining experiments and computational models, we show that the morphological properties of muscles allow them to minimize the number of slow intercellular signaling steps and act as bidirectional superhighways for propagating wound signals and instructing responses in other cell types. Inhibiting Erk propagation prevents cells distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues within a narrow time window after the first injury. These results suggest that rapid responses in uninjured tissues far from wounds are essential for regeneration. Our findings provide a mechanism for long-range signal propagation in large and complex tissues to coordinate cellular responses across diverse cell types, and highlights the function of feedback between spatially separated tissues during whole-body regeneration.

Adaptive robustness through incoherent signaling mechanisms in a regenerative brain.

Animal behavior emerges from collective dynamics of interconnected neurons, making it vulnerable to connectome damage. Paradoxically, many organisms maintain significant behavioral output after large-scale neural injury. Molecular underpinnings of this extreme robustness remain largely unknown. Here, we develop a quantitative behavioral analysis pipeline to measure previously uncharacterized long-lasting latent memory states in planarian flatworms during whole-brain regeneration. By combining >20,000 animal trials with neural population dynamic modeling, we show that long-range volumetric peptidergic signals allow the planarian to rapidly reestablish latent states and restore coarse behavior after large structural perturbations to the nervous system, while small-molecule neuromodulators gradually refine the precision. The different time and length scales of neuropeptide and small-molecule transmission generate incoherent patterns of neural activity which competitively regulate behavior and memory. Controlling behavior through opposing communication mechanisms creates a more robust system than either alone and may serve as a generic approach to construct robust neural networks.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.

Emergency airway management in a Singapore centre: A registry study.

INTRODUCTION: Intubations in the emergency department (ED) are often performed immediately without the benefit of pre-selection or the ability to defer. Multicentre observational data provide a framework for understanding emergency airway management but regional practice variation may exist. We aim to describe the intubation indications, prevalence of difficult airway features, peri-intubation adverse events and intubator characteristics in the ED of the National University Hospital, Singapore. METHODS: We conducted a prospective observational study over a period of 31 months from 1 March 2016 to 28 September 2018. Information regarding each intubation attempt, such as indications for intubation, airway assessment, intubation techniques used, peri-intubation adverse events, and clinical outcomes, was collected and described. RESULTS: There were 669 patients, with male predominance (67.3%, 450/669) and mean age of 60.9 years (standard deviation [SD] 18.1). Of these, 25.6% were obese or grossly obese and majority were intubated due to medical indications (84.8%, 567/669). Emergency physicians' initial impression of difficult airway correlated with a higher grade of glottis view on laryngoscopy. First-pass intubation success rate was 86.5%, with hypoxia (11.2%, 75/669) and hypotension (3.7%, 25/669) reported as the two most common adverse events. Majority was rapid sequence intubation (67.3%, 450/669) and the device used was most frequently a video laryngoscope (75.6%, 506/669). More than half of the intubations were performed by postgraduate clinicians in year 5 and above, clinical fellows or attending physicians. CONCLUSION: In our centre, the majority of emergency intubations were performed for medical indications by senior doctors utilising rapid sequence intubation and video laryngoscopy with good ffirst-attempt success.

Management of acute pyelonephritis in the emergency department observation unit.

INTRODUCTION: This study aimed to assess the effectiveness of the emergency department observation unit (EDOU) for patients with acute pyelonephritis in a Singapore tertiary academic medical centre. METHODS: We reviewed the clinical records of consecutive patients who presented with pyelonephritis between 1 July 2012 and 31 October 2014 to collect information on demographics, symptoms, signs, laboratory and radiological results, treatment, and clinical outcomes. RESULTS: Of 459 emergency department (ED) patients who were identified as having pyelonephritis, 164 (35.7%) were managed in the EDOU. Successful management in the EDOU was achieved in 100 (61.0%) patients. Escherichia coli was the predominant (64.6%) micro-organism in urine cultures and was positive in 106 patients. Patients diagnosed with acute pyelonephritis who were successfully managed in the EDOU had a lower incidence of nausea (32.0% vs. 60.9%, p < 0.001) and vomiting (15.0% vs. 50.0%, p < 0.001) compared to those who were not successful. CONCLUSION: EDOU is useful for both observation and treatment of patients with acute pyelonephritis. Urine cultures are sufficient for the identification of the culprit micro-organism. Patients who present with prominent symptoms of vomiting should have routine administration of antiemetics, while consideration for second-line antiemetics is recommended for those with persistent symptoms.

A deep-learning system for the assessment of cardiovascular disease risk via the measurement of retinal-vessel calibre.

Retinal blood vessels provide information on the risk of cardiovascular disease (CVD). Here, we report the development and validation of deep-learning models for the automated measurement of retinal-vessel calibre in retinal photographs, using diverse multiethnic multicountry datasets that comprise more than 70,000 images. Retinal-vessel calibre measured by the models and by expert human graders showed high agreement, with overall intraclass correlation coefficients of between 0.82 and 0.95. The models performed comparably to or better than expert graders in associations between measurements of retinal-vessel calibre and CVD risk factors, including blood pressure, body-mass index, total cholesterol and glycated-haemoglobin levels. In retrospectively measured prospective datasets from a population-based study, baseline measurements performed by the deep-learning system were associated with incident CVD. Our findings motivate the development of clinically applicable explainable end-to-end deep-learning systems for the prediction of CVD on the basis of the features of retinal vessels in retinal photographs.

Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells.

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infections, employing numerous molecular strategies to contribute to adhesion, colonization, and persistence in the bladder niche. Identifying strategies to prevent adhesion and colonization is a promising approach to inhibit bacterial pathogenesis and to help preserve the efficacy of available antibiotics. This approach requires an improved understanding of the molecular determinants of adhesion to the bladder urothelium. We designed experiments using a custom-built live cell monolayer rheometer (LCMR) to quantitatively measure individual and combined contributions of bacterial cell surface structures [type 1 pili, curli, and phosphoethanolamine (pEtN) cellulose] to bladder cell adhesion. Using the UPEC strain UTI89, isogenic mutants, and controlled conditions for the differential production of cell surface structures, we discovered that curli can promote stronger adhesive interactions with bladder cells than type 1 pili. Moreover, the coproduction of curli and pEtN cellulose enhanced adhesion. The LCMR enables the evaluation of adhesion under high-shear conditions to reveal this role for pEtN cellulose which escaped detection using conventional tissue culture adhesion assays. Together with complementary biochemical experiments, the results support a model wherein cellulose serves a mortar-like function to promote curli association with and around the bacterial cell surface, resulting in increased bacterial adhesion strength at the bladder cell surface.

Management of dyspepsia-The role of the ED Observation unit to optimize patient outcomes.

BACKGROUND: Dyspepsia is a common complaint that can confer significant burden on one's quality of life and may also be associated with serious underlying conditions. The objective of this study was to determine if patients admitted to the emergency department observation unit (EDOU) for severe or persistent dyspepsia would have cost effective management in terms of investigations performed, length and cost of hospital stay. The secondary objective was to determine if any patient characteristics could predict a need for admission to the inpatient unit. METHODS: Retrospective chart reviews of patients admitted to the EDOU under the Dyspepsia protocol between January 2008 and August 2014 were conducted. Baseline demographics, investigations performed, outcomes related to EDOU stay, admission and 30-day re-presentation outcomes were recorded. RESULTS: A total of 1304 patients were included. Median length of stay was 1day. Cumulative bed-saved days were 38 per month. Two hundred eighteen (16.7%) patients required admission to the inpatient service for further management, while 533 (40.9%) and 313 (24.0%) patients underwent esophagogastroduodenoscopy and hepatobiliary ultrasonography, respectively. No major adverse events were attributed to the EDOU admissions or delays in treatment. No significant clinically relevant factors were associated with a need for admission from the EDOU to the inpatient unit. Median cost of the EDOU admission was approximately one-third that of a similar admission to the inpatient unit. CONCLUSION: The EDOU is an appropriate setting to facilitate investigations and treatment of patients with dyspepsia with considerable bed-saved days.

Interfacial mechanisms for stability of surfactant-laden films.

Thin liquid films are central to everyday life. They are ubiquitous in modern technology (pharmaceuticals, coatings), consumer products (foams, emulsions) and also serve vital biological functions (tear film of the eye, pulmonary surfactants in the lung). A common feature in all these examples is the presence of surface-active molecules at the air-liquid interface. Though they form only molecular-thin layers, these surfactants produce complex surface stresses on the free surface, which have important consequences for the dynamics and stability of the underlying thin liquid film. Here we conduct simple thinning experiments to explore the fundamental mechanisms that allow the surfactant molecules to slow the gravity-driven drainage of the underlying film. We present a simple model that works for both soluble and insoluble surfactant systems in the limit of negligible adsorption-desorption dynamics. We show that surfactants with finite surface rheology influence bulk flow through viscoelastic interfacial stresses, while surfactants with inviscid surfaces achieve stability through opposing surface-tension induced Marangoni flows.

Instability and Breakup of Model Tear Films.

PURPOSE: An experimental platform to replicate the human tear film on a contact lens is presented. The influence of interfacial viscoelasticity in stabilizing in vitro model tear films against breakup and dewetting is investigated using this instrument. METHODS: Model tear films consisting of bovine meibomian lipids (meibum) spread on either PBS or artificial tear solution (ATS) are created. The interfacial shear rheology of these films is measured as a function of temperature. The dewetting dynamics of these films is then investigated using the Interfacial Dewetting and Drainage Optical Platform (i-DDrOP) on top of silicone hydrogel (SiHy) contact lenses at 23 and 35°C. The film breakup times are evaluated using two parameters: onset of film breakup, Tonset for thick films (∼100 µm), and tear breakup times, TBU for thin films (∼1 µm). Thin film thinning rates as a result of evaporation are also calculated. RESULTS: The ATS/meibum films have the largest surface rheology and correspondingly show the largest Tonset times at both 23 and 35°C. The parameter TBU is also significantly larger for ATS/meibum (TBU ∼ 40 seconds) compared with that of ATS and PBS/meibum films (TBU ∼ 30 seconds) at room temperature. However, at 35°C, all three model tear films exhibit similar TBU ∼ 17 seconds and average rate of thinning of -4 µm/minute. CONCLUSIONS: Tear film stability is influenced by both surface rheology and evaporation. The in vitro tear breakup times and thinning rates of model tear films at 35°C are in good agreement with in vivo measurements previously reported, highlighting the utility of the i-DDrOP for in vitro tear film breakup research.