The microenvironment dictates glycocalyx construction and immune surveillance.

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.

Tissue tension permits ß-catenin phosphorylation to drive mesoderm specification in human embryonic stem cells.

The role of morphogenetic forces in cell fate specification is an area of intense interest. Our prior studies suggested that the development of high cell-cell tension in human embryonic stem cells (hESC) colonies permits the Src-mediated phosphorylation of junctional ß-catenin that accelerates its release to potentiate Wnt-dependent signaling critical for initiating mesoderm specification. Using an ectopically expressed nonphosphorylatable mutant of ß-catenin (Y654F), we now provide direct evidence that impeding tension-dependent Src-mediated ß-catenin phosphorylation impedes the expression of Brachyury (T) and the epithelial-to-mesenchymal transition (EMT) necessary for mesoderm specification. Addition of exogenous Wnt3a or inhibiting GSK3ß activity rescued mesoderm expression, emphasizing the importance of force dependent Wnt signaling in regulating mechanomorphogenesis. Our work provides a framework for understanding tension-dependent ß-catenin/Wnt signaling in the self-organization of tissues during developmental processes including gastrulation.

Mechanical Tension Promotes Formation of Gastrulation-like Nodes and Patterns Mesoderm Specification in Human Embryonic Stem Cells.

Embryogenesis is directed by morphogens that induce differentiation within a defined tissue geometry. Tissue organization is mediated by cell-cell and cell-extracellular matrix (ECM) adhesions and is modulated by cell tension and tissue-level forces. Whether cell tension regulates development by modifying morphogen signaling is less clear. Human embryonic stem cells (hESCs) exhibit an intrinsic capacity for self-organization, which motivates their use as a tractable model of early human embryogenesis. We engineered patterned substrates that recapitulate the biophysical properties of the early embryo and mediate the self-organization of "gastrulation-like" nodes in cultured hESCs. Tissue geometries that generated local nodes of high cell-adhesion tension directed the spatial patterning of the BMP4-dependent "gastrulation-like" phenotype by enhancing phosphorylation and junctional release of ß-catenin to promote Wnt signaling and mesoderm specification. Furthermore, direct force application via mechanical stretching promoted BMP-dependent mesoderm specification, confirming that tissue-level forces can directly regulate cell fate specification in early human development.

Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.

Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein ß-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.

Challenges in the Regulation of High-Cost Treatments: An Overview From Brazil.

Regulation of drug prices that ensures adequate access to effective treatments and promotes innovation is a global challenge. In the United States, the government does not regulate drug prices when they come onto market. On the other hand, in countries such as France and Brazil, government agencies are responsible for setting up price limits by leveraging the interests of the companies and the countries' population. In Brazil, safety and efficacy of drugs are regulated by the Brazilian Health Regulatory Agency, and drug prices are regulated by the Pharmaceutical Market Regulation Chamber with a participation of Brazilian Health Regulatory Agency. Here, we introduce the current challenges faced by the Brazilian government in the drug price regulation and present proposed initiatives aiming to streamline access to innovative treatments for its citizens.

Tissue mechanics, an important regulator of development and disease.

A growing body of work describes how physical forces in and around cells affect their growth, proliferation, migration, function and differentiation into specialized types. How cells receive and respond biochemically to mechanical signals is a process termed mechanotransduction. Disease may arise if a disruption occurs within this mechanism of sensing and interpreting mechanics. Cancer, cardiovascular diseases and developmental defects, such as during the process of neural tube formation, are linked to changes in cell and tissue mechanics. A breakdown in normal tissue and cellular forces activates mechanosignalling pathways that affect their function and can promote disease progression. The recent advent of high-resolution techniques enables quantitative measurements of mechanical properties of the cell and its extracellular matrix, providing insight into how mechanotransduction is regulated. In this review, we will address the standard methods and new technologies available to properly measure mechanical properties, highlighting the challenges and limitations of probing different length-scales. We will focus on the unique environment present throughout the development and maintenance of the central nervous system and discuss cases where disease, such as brain cancer, arises in response to changes in the mechanical properties of the microenvironment that disrupt homeostasis. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Computational fluid dynamic analysis of physical forces playing a role in brain organoid cultures in two different multiplex platforms.

BACKGROUND: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined. RESULTS: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask. CONCLUSION: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.

Weaving for heart valve tissue engineering.

Weaving is a resourceful technology which offers a large selection of solutions that are readily adaptable for tissue engineering (TE) of artificial heart valves (HV). The different ways that the yarns are interlaced in this technique could be used to produce complex architectures, such as the three-layer architecture of the leaflets. Once the assembly is complete, growth of cells in the scaffold would occur in the orientation of the yarn, enabling the deposition of extra cellular matrixes proteins in an oriented manner. Weaving technology is a rapidly evolving field that, first, needs to be understood, and then explored by tissue engineers, so that it could be used to create efficient scaffolds. Similarly, the textile engineers need to gain a basic understanding of key structural and mechanical aspects of the heart valve. The aim of this review is to provide the platform for joining these two fields and to enable cooperative research efforts. Moreover, examples of woven medical products and patents as well as related publication are discussed in this review, nevertheless due to the large, and continuously growing volume of data, only the aspects strictly associated with HVTE lay in the scope of this paper.

Valve Tissue Engineering with Living Absorbable Threads.

Tissue engineering (TE) depends on the population of scaffolds with appropriate cells, arranged in a specific physiological direction using a variety of techniques. Here, a novel technique of creating "living threads" is described based on thin (poly(ε-caprolactone) fibers of different diameters (23-243 µm). The fibers readily attract human mesenchymal stem cells (MSCs), which are firmly adhered. These versatile fibers can be used to produce dimensional shapes identical in shape to the cup-like structure of a normal human valve, while preserving the specific orientation of both the cells and the fibers. The MSCs on leaflets and the cells cultured in flask shown similar epitopes expression when analyzed by fluorescence activated cell sorting. Together, these characteristics have important functional implications as living absorbable fibers can be a valuable resource in TE of living tissues, including heart valves.

Knitting for heart valve tissue engineering.

Knitting is a versatile technology which offers a large portfolio of products and solutions of interest in heart valve (HV) tissue engineering (TE). One of the main advantages of knitting is its ability to construct complex shapes and structures by precisely assembling the yarns in the desired position. With this in mind, knitting could be employed to construct a HV scaffold that closely resembles the authentic valve. This has the potential to reproduce the anisotropic structure that is characteristic of the heart valve with the yarns, in particular the 3-layered architecture of the leaflets. These yarns can provide oriented growth of cells lengthwise and consequently enable the deposition of extracellular matrix (ECM) proteins in an oriented manner. This technique, therefore, has a potential to provide a functional knitted scaffold, but to achieve that textile engineers need to gain a basic understanding of structural and mechanical aspects of the heart valve and in addition, tissue engineers must acquire the knowledge of tools and capacities that are essential in knitting technology. The aim of this review is to provide a platform to consolidate these two fields as well as to enable an efficient communication and cooperation among these two research areas.

Limited diagnostic value of Wells-score and D-dimer testing in hospitalized dermatologic patients with symptoms of deep vein thrombosis.

BACKGROUND: D-dimer analysis and clinical probability scoring (Wells-score) show a high sensitivity and negative predictive value for the exclusion of deep vein thrombosis (DVT). OBJECTIVE: To identify the diagnostic performance of D-dimer testing and Wells-score in hospitalized patients with dermatologic conditions. METHODS: In this retrospective cohort study, 109 examinations in 102 patients were performed by Wells-score, Tina-quant D-dimer testing and whole-leg duplex ultrasonography or phlebography. RESULTS: DVT was confirmed in 14 patients. The Wells-score alone allowed no discrimination of DVT and non-DVT patients. D-dimer testing identified all cases of DVT (100% sensitivity). Only 16 patients showed D-dimers within normal limits and none was diagnosed with DVT (100% negative predictive value). A high rate of false-positive D-dimer results (72%) led to a low specificity (17%). The number needed-to-test to exclude one DVT was 6.8. Based on multivariate statistical analysis, increased D-dimer levels were significantly associated with the dermatologic main diagnosis (p = 0.008), age (p = 0.001) and with the presence of DVT (p = 0.011). The highest D-dimer values were found in non-DVT patients with metastasized or systemic malignancies (median 2.48 mg/L) or inflammatory skin conditions (e.g., generalized psoriasis, median 2.22 mg/L). CONCLUSIONS: Wells-score and D-Dimer testing were of limited diagnostic value because of many false-positive results. Required imaging procedures were reduced by only 16 cases (15%). Therefore, we suggest directly investigating hospitalized dermatologic patients with suspected DVT and skin diseases associated with high D-Dimer levels, by whole-leg compression ultrasonography.