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Dense and aligned Collagen I fibers are associated with collective cancer invasion led by protrusive tumor cells, leader cells. In some breast tumors, a population of cancer cells (basal-like cells) maintain several epithelial characteristics and express the myoepithelial/basal cell marker Keratin 14 (K14). Emergence of leader cells and K14 expression are regarded as interconnected events triggered by Collagen I, however the underlying mechanisms remain unknown. Using breast carcinoma organoids, we show that Collagen I drives a force-dependent loop, specifically in basal-like cancer cells. The feed-forward loop is centered around the mechanotransducer Yap and independent of K14 expression. Yap promotes a transcriptional program that enhances Collagen I alignment and tension, which further activates Yap. Active Yap is detected in invading breast cancer cells in patients and required for collective invasion in 3D Collagen I and in the mammary fat pad of mice. Our work uncovers an essential function for Yap in leader cell selection during collective cancer invasion. Aligned collagen I is associated with the emergence of leader cells that are responsible for collective invasion. Here, the authors show that Collagen I and Yap signalling are in a feed-forward loop to drive the collective invasion of basal-like tumour cells.

The tumor micro-environment often contains stiff and irregular-bundled collagen fibers that are used by tumor cells to disseminate. It is still unclear how and to what extent, extracellular matrix (ECM) stiffness versus ECM bundle size and alignment dictate cancer cell invasion. Here, we have uncoupled Collagen-I bundling from stiffness by introducing inter-collagen crosslinks, combined with temperature induced aggregation of collagen bundling. Using organotypic models from mouse invasive ductal and invasive lobular breast cancers, we show that increased collagen bundling in 3D induces a generic increase in breast cancer invasion that is independent of migration mode. However, systemic collagen stiffening using advanced glycation end product (AGE) crosslinking prevents collective invasion, while leaving single cell invasion unaffected. Collective invasion into collagen matrices by ductal breast cancer cells depends on Lysyl oxidase-like 3 (Loxl3), a factor produced by tumor cells that reinforces local collagen stiffness. Finally, we present clinical evidence that collectively invading cancer cells at the invasive front of ductal breast carcinoma upregulate LOXL3. By uncoupling the mechanical, chemical, and structural cues that control invasion of breast cancer in three dimensions, our data reveal that spatial control over stiffness and bundling underlie collective dissemination of ductal-type breast cancers.

CRYAB (αB-crystallin) is expressed in many tissues and yet the R120G mutation in CRYAB causes tissue-specific pathologies, namely cardiomyopathy and cataract. Here, we present evidence to demonstrate that there is a specific functional interaction of CRYAB with desmin intermediate filaments that predisposes myocytes to disease caused by the R120G mutation. We use a variety of biochemical and biophysical techniques to show that plant, animal and ascidian small heat-shock proteins (sHSPs) can interact with intermediate filaments. Nevertheless, the mutation R120G in CRYAB does specifically change that interaction when compared with equivalent substitutions in HSP27 (R140G) and into the Caenorhabditis elegans HSP16.2 (R95G). By transient transfection, we show that R120G CRYAB specifically promotes intermediate filament aggregation in MCF7 cells. The transient transfection of R120G CRYAB alone has no significant effect upon cell viability, although bundling of the endogenous intermediate filament network occurs and the mitochondria are concentrated into the perinuclear region. The combination of R120G CRYAB co-transfected with wild-type desmin, however, causes a significant reduction in cell viability. Therefore, we suggest that while there is an innate ability of sHSPs to interact with and to bind to intermediate filaments, it is the specific combination of desmin and CRYAB that compromises cell viability and this is potentially the key to the muscle pathology caused by the R120G CRYAB.

Fibrin is the major extracellular component of blood clots and a proteinaceous hydrogel used as a versatile biomaterial. Fibrin forms branched networks of polymeric fibers, built of laterally associated double-stranded protofibrils. This multiscale hierarchical structure is crucial for the extraordinary mechanical resilience of blood clots. Yet, the structural basis of clot mechanical properties remains largely unclear due, in part, to the unresolved molecular packing structure of fibrin fibers. Here we quantitatively assess the packing structure of fibrin fibers by combining Small Angle X-ray Scattering (SAXS) measurements of fibrin networks reconstituted under a wide range of conditions with computational molecular modeling of fibrin oligomers. The number, positions, and intensities of the Bragg peaks observed in the SAXS experiments were reproduced computationally based on the all-atom molecular structure of reconstructed fibrin protofibrils. Specifically, the model correctly predicts the intensities of the reflections of the 22.5 nm axial repeat, corresponding to the half-staggered longitudinal arrangement of fibrin molecules. In addition, the SAXS measurements showed that protofibrils within fibrin fibers have a partially ordered lateral arrangement with a characteristic transverse repeat distance of 13 nm, irrespective of the fiber thickness. These findings provide fundamental insights into the molecular structure of fibrin clots that underlies their biological and physical properties.

Objective To compare the diagnostic accuracy of TE and MRE and establish cutoff levels and diagnostic strategies for both techniques, enabling selection of patients for liver biopsy. Methods One hundred three patients with chronic hepatitis B or C and liver biopsy were prospectively included. Areas under curves (AUROC) were compared for TE and MRE for METAVIR fibrosis grade ≥ F2 and ≥F3. We defined cutoff values for selection of patients with F0–F1 (sensitivity >95 %) and for significant fibrosis F2–F4 (specificity >95 %). Results Following exclusions, 85 patients were analysed (65 CHB, 19 CHC, 1 co-infected). Fibrosis stages were F0 ( n  = 3), F1 ( n  = 53), F2 ( n  = 15), F3 ( n  = 8) and F4 ( n  = 6). TE and MRE accuracy were comparable [AUROC TE  ≥ F2: 0.914 (95 % CI: 0.857–0.972) vs. AUROC MRE  ≥ F2: 0.909 (0.840–0.977), P  = 0.89; AUROC TE  ≥ F3: 0.895 (0.816–0.974) vs. AUROC MRE  ≥ F3: 0.928 (0.874–0.982), P  = 0.42]. Cutoff values of <5.2 and ≥8.9 kPa (TE) and <1.66 and ≥2.18 kPa (MRE) diagnosed 64 % and 66 % of patients correctly as F0–F1 or F2–F4. A conditional strategy in inconclusive test results increased diagnostic yield to 80 %. Conclusion TE and MRE have comparable accuracy for detecting significant fibrosis, which was reliably detected or excluded in two-thirds of patients. A conditional strategy further increased diagnostic yield to 80 %. Key Points • Both ultrasound-based transient elastography and magnetic resonance elastography can assess hepatic fibrosis. • Both have comparable accuracy for detecting liver fibrosis in viral hepatitis. • The individual techniques reliably detect or exclude significant liver fibrosis in 66 %. • A conditional strategy for inconclusive findings increases the number of correct diagnoses.

Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or ‘tubuloids’. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function.‘Tubuloids’ grown from human kidney tissue and urine aid the study of BK virus infection, Wilms tumors and cystic fibrosis.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8 + resident-memory (T RM ) and CD4 + T-helper-17 (T H17 ) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4 + T-cells with T-helper-1 characteristics (T H1 -like) and CD8 + T-cells expressing exhaustion markers (T EX -like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Background Mental health problems often arise in childhood and adolescence and can have detrimental effects on people’s quality of life (QoL). Therefore, it is of great importance for clinicians, policymakers and researchers to adequately measure QoL in children. With this review, we aim to provide an overview of existing generic measures of QoL suitable for economic evaluations in children with mental health problems. Methods First, we undertook a meta-review of QoL instruments in which we identified all relevant instruments. Next, we performed a systematic review of the psychometric properties of the identified instruments. Lastly, the results were summarized in a decision tree. Results This review provides an overview of these 22 generic instruments available to measure QoL in children with psychosocial and or mental health problems and their psychometric properties. A systematic search into the psychometric quality of these instruments found 195 suitable papers, of which 30 assessed psychometric quality in child and adolescent mental health. Conclusions We found that none of the instruments was perfect for use in economic evaluation of child and adolescent mental health care as all instruments had disadvantages, ranging from lack of psychometric research, no proxy version, not being suitable for young children, no age-specific value set for children under 18, to insufficient focus on relevant domains (e.g. social and emotional domains).