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Responsive nanomaterials that can undergo reversible changes in morphology are interesting for the development of functional materials that interact with and respond to their environment. Amphiphilic block copolymers are well-known for their ability to create a wide range of supramolecular nanostructures in solution. Arylazopyrazoles (AAPs) are versatile molecular photoswitches, which change their configuration and hydrophobicity upon irradiation with UV light (365 nm, Z isomer, less hydrophobic) and green light (520 nm, E isomer, more hydrophobic). In this work, photoswitchable block copolymers containing arylazopyrazole tetraethylene glycol methacrylate (AAPMA) and oligo(ethylene glycol) methacrylate (OEGMA) forming amphiphilic POEGMA- b -PAAPMA with varying block lengths are prepared by RAFT polymerization. The photochemical properties of AAP persist in the polymers. Due to their amphiphilic structure, the polymers self-assemble into supramolecular morphologies in water. Remarkably, photoisomerization results in a reversible change in the self-assembly behavior. Specifically, spherical and cylindrical micelles are observed for POEGMA 33 - b -PAAPMA 47 when illuminated with green or UV light during assembly. Furthermore, the morphology of assembled structures can be reversibly switched by subsequent irradiation with UV and green light.

This study investigates unexpected competitive host-guest interactions of β-cyclodextrin (β-CD), which can occur with polymers in aqueous solution, using the examples of the two polymers poly(oligo(ethylene glycol) methyl ether methacrylate) and poly(glycerol mono methacrylate). Systematic structural modifications of the polymer provide insight into the host-guest interaction with β-CD and the competition between side chains and end groups such as hydrophobic end groups remaining from reversible addition fragmentation chain transfer polymerization or intentionally implemented molecular recognition units such as arylazopyrazole photoswitches., (© 2024 Wiley‐VCH GmbH.)

Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo . These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics., Competing Interests: All authors are current or former full-time employees and shareholders of Surrozen, Inc. Y.L. is Executive Vice President of Research at Surrozen, Inc. Patent applications are pending for the work described in this manuscript., (© 2024 The Author(s).)

Background: Wnt/β-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies., Methods: Using our antibody-based platform of Wnt/β-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures., Results: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo., Conclusions: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/β-catenin pathway modulation for the treatment of pulmonary fibrosis., (© 2024. The Author(s).)

Systemic toxicity is a major challenge in the development of therapeutics. Consequently, cell-type-specific targeting is needed to improve on-target efficacy while reducing off-target toxicity. Here, we describe a cell-targeting system we have termed BRAID ( BR idged A ctivation by I ntra/intermolecular D ivision) whereby an active molecule is divided into two inactive or less active parts that are subsequently brought together via a so-called 'bridging receptor' on the target cell. This concept was validated using the WNT/β-catenin signaling system, demonstrating that a multivalent WNT agonist molecule divided into two inactive components assembled from different epitopes via the hepatocyte receptor βKlotho induces signaling specifically on hepatocytes. These data provide proof of concept for this cell-specific targeting strategy, and in principle, this may also allow activation of multiple signaling pathways where desirable. This approach has broad application potential for other receptor systems., Competing Interests: HC, SL, NS, TS, CL, YP The authors are current full-time employees and shareholders of Surrozen, Inc, RL, AS, AD, YP, MV The authors were former full-time employees and shareholders of Surrozen, Inc, YL The author is a current full-time employee and shareholder of Surrozen, Inc. YL is Executive Vice President of Research at Surrozen, Inc, (© 2023, Chen, Lee et al.)

The synthesis and properties of Janus nanoparticles with spherical, cylindrical, and disk-like shapes are nowadays rather well understood. Other topologies such as nanorings and bowl-shaped Janus nanoparticles are believed to show distinctly different solution behavior and interaction with interfaces, but limitations in their synthesis currently prevents a proper investigation of these properties. Especially the combination of shape- and surface-anisotropy of bowl-shaped Janus nanoparticles could result in enhanced selectivity in uptake of cargo and enhanced directional diffusion. We here produce bowl-shaped Janus nanoparticles without noticeable side products through evaporation-induced confinement assembly (EICA) of triblock terpolymers blended with high molecular weight homopolymer. The triblock terpolymer phase separates from the homopolymer into spherical domes, where the terpolymer adopts a hemispherical lamella-lamella morphology ( ll ). Selective cross-linking, removal of the homopolymer, and disassembly of the microparticles releases the bowl-shaped Janus nanoparticles. The amount of blended homopolymer determines the size of the spherical dome, allowing to control particle curvature into flat Janus nanoplates, hemispherical Janus nanobowls, and deep Janus nanocups. The use of Shirasu Porous Glass (SPG) membranes with pore sizes in the range of d pore = 0.2-2.0 μm further provides control of particle diameter. Size and shape were analyzed with electron microscopy and the Janus character through selective surface decoration. The diffusion behavior of bowl-shaped Janus nanoparticles was investigated depending on particle curvature and anisotropy using angle-dependent dynamic light scattering.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/β-catenin-activating Frizzleds (FZDs). We show that FZD 1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development., Competing Interests: Declaration of interests All authors are current or former full-time employees and shareholders of Surrozen, Inc. W.C.Y. is Chief Scientific Officer and Y.L. is Senior Vice President, Biology at Surrozen, Inc. A patent application is pending for the work described in this manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland., Competing Interests: Declaration of interests H.C. is the inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. Y.P. is an employee of Surrozen and was an employee of the Hubrecht Institute when this work was performed and completed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Classic studies on hematopoiesis indicate that blood cell numbers are maintained by rare, hard-wired, transplantable stem cells (SCs). Subsequent studies in other organs have implicitly assumed that all SC hierarchies follow the design of the hematopoietic system. Lineage tracing techniques have revolutionized the study of solid tissue SCs. It thus appears that key characteristics of the hematopoietic SC hierarchy (rarity of SCs, specific marker expression, quiescence, asymmetric division, and unidirectional differentiation) are not generalizable to other tissues. In light of these insights, we offer a revised, generalizable definition of SC function: the ability to replace lost tissue through cell division., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Patients from opioid maintenance treatment (OMT) may taper off from opioids during rehab treatment. It is investigated whether this option improves treatment outcomes in particular for those patients who are affected by psychiatric or somatic comorbidity, or by social problems., Methods: Multicenter study with 4 rehab units. Patient characteristics and treatment outcomes were collected retrospectively. There were n = 227 patients who were admitted to treatment while still receiving OMT, and n = 156 who were opioid free., Results: With regard to regular completion of rehab treatment, an interaction was observed between status at admission and diagnosis of affective, neurotic and anxiety disorders, and between status and previous rehab treatment experience. If admitted in an abstinent status, patients who had never entered rehab, and patients with affective or anxiety disorders showed decreased regular completion rates, while they showed increased completion rates if tapered off during treatment. There was no interaction between initial status and acute life crisis (release from prison, homelessness), somatic diagnoses, or number of stabilizing social factors., Conclusion: Admission while still receiving opiate maintenance could be helpful for patients with psychiatric comorbidity and for patients who had previously been reluctant to enter rehab treatment., Competing Interests: Norbert Scherbaum hat für Tätigkeiten in Advisory Boards, Vortragstätigkeit, Erstellung von Manuskripten, Erstellung von Weiterbildungsmaterial Honorare der Firmen AbbVie, Hexal, Janssen-Cielag, Lundbeck, MSD, Medice, Munidpharma, Reckitt-Benckiser/Indivior und Sanofi-Aventis erhalten. Er hat in den letzten 3 Jahren an Medikamentenprüfungen teilgenommen, die von der pharmazeutischen Industrie finanziert wurden. Die übrigen Autoren geben keine möglichen Interessenkonflikte an., (© Georg Thieme Verlag KG Stuttgart · New York.)

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of Fstl1 in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury., Competing Interests: Conflict of interest statement: H.C. is co-principal investigator (co-PI) on a Dutch grant on organs-on-a-chip with C.L.M. as co-PI, however, on an unrelated topic., (Copyright © 2018 the Author(s). Published by PNAS.)

Enteroendocrine cells (EECs) control a wide range of physiological processes linked to metabolism 1 . We show that EEC hormones are differentially expressed between crypts (for example, Glp1) and villi (for example, secretin). As demonstrated by single-cell mRNA sequencing using murine Lgr5 + cell-derived organoids, BMP4 signals alter the hormone expression profiles of individual EECs to resemble those found in the villus. Accordingly, BMP4 induces hormone switching of EECs migrating up the crypt-villus axis in vivo. Our findings imply that EEC lineages in the small intestine exhibit a more flexible hormone repertoire than previously proposed. We also describe a protocol to generate human EECs in organoids and demonstrate a similar regulation of hormone expression by BMP signalling. These findings establish alternative strategies to target EECs with therapeutically relevant hormone production through BMP modulation.

Here we describe a simple method to measure larval muscle contraction and locomotion behavior. The method enables the user to acquire data, without the necessity of using expensive equipment ( Rotstein et al. , 2018 ). To measure contraction and locomotion behaviour, single larvae are positioned at the center of a humidified Petri dish. Larval movement is recorded over time using the movie function of a consumer digital camera. Subsequently, videos are analyzed using ImageJ ( Rueden et al. , 2017 ) for distance measurements and counting of contractions. Data are represented as box or scatter plots using GraphPad Prism ( © GraphPad Software)., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

The biomechanical properties of extracellular matrices (ECMs) are critical to many biological processes, including cell-cell communication and cell migration and function. The correct balance between stiffness and elasticity is essential to the function of numerous tissues, including blood vessels and the lymphatic system, and depends on ECM constituents (the "matrisome") and on their level of interconnection. However, despite its physiological relevance, the matrisome composition and organization remain poorly understood. Previously, we reported that the ADAMTS-like protein Lonely heart (Loh) is critical for recruiting the type IV collagen-like protein Pericardin to the cardiac ECM. Here, we utilized Drosophila as a simple and genetically amenable invertebrate model for studying Loh-mediated recruitment of tissue-specific ECM components such as Pericardin to the ECM. We focused on the functional relevance of distinct Loh domains to protein localization and Pericardin recruitment. Analysis of Loh deletion constructs revealed that one thrombospondin type 1 repeat (TSR1-1), which has an embedded W XX W motif, is critical for anchoring Loh to the ECM. Two other thrombospondin repeats, TSR1-2 and TSR1-4, the latter containing a C XX TC XX G motif, appeared to be dispensable for tethering Loh to the ECM but were crucial for proper interaction with and recruitment of Pericardin. Moreover, our results also suggested that Pericardin in the cardiac ECM primarily ensures the structural integrity of the heart, rather than increasing tissue flexibility. In conclusion, our work provides new insights into the roles of thrombospondin type 1 repeats and advances our understanding of cardiac ECM assembly and function., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

A rational-based physical descriptive model (PDM) has been developed to predict the levels of Escherichia coli in water at a beach with dynamic conditions in the Greater Toronto Area (GTA), Ontario, Canada. Bacteria loadings in the water were affected not only by multiple physical factors (precipitation, discharge, wind, etc.), but also by cumulative effects, intensity, duration and timing of storm events. These may not be linearly related to the observed variations in bacteria levels, and are unlikely to be properly represented by a widely used multiple linear regression model. In order to account for these complex relationships, the amounts of precipitation and nearby creek discharge, the impact of various time-related factors, lag time between events and sample collection, and threshold for different parameters were used in determining bacteria levels. This new comprehensive PDM approach improved the accuracy of the E. coli level predictions in the studied beach water compared to the previously developed statistical predictive and presently used geometric mean models. In spite of the complexity and dynamic conditions at the studied beach, the PDM achieved 75% accuracy overall for the five case years examined.

Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells., (Copyright © 2015, American Association for the Advancement of Science.)

Objective measures of nonspeech orofacial strength and force control were obtained for 20 individuals with traumatic brain injury. The dynamic and static force generating abilities of the upper lip, lower lip, tongue, and jaw were assessed. Based on sentence intelligibility scores, the subjects were divided into two groups, more and less intelligible. Force measures included reaction time, slope, derivative, peak overshoot, and first- and second-half mean hold and standard deviation. Groups differed only in the ability to sustain the 2 N force level with the tongue. Other potential contributors to the differences in intelligibility are discussed.