Integrating bioengineering, super-resolution microscopy and mechanobiology in autophagy research: addendum to the guidelines (4th edition).

Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell. Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy.

Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging.

Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogs were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm-specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

VolumePeeler: a novel FIJI plugin for geometric tissue peeling to improve visualization and quantification of 3D image stacks.

MOTIVATION: Quantitative descriptions of multi-cellular structures from optical microscopy imaging are prime to understand the variety of three-dimensional (3D) shapes in living organisms. Experimental models of vertebrates, invertebrates and plants, such as zebrafish, killifish, Drosophila or Marchantia, mainly comprise multilayer tissues, and even if microscopes can reach the needed depth, their geometry hinders the selection and subsequent analysis of the optical volumes of interest. Computational tools to "peel" tissues by removing specific layers and reducing 3D volume into planar images, can critically improve visualization and analysis. RESULTS: We developed VolumePeeler, a versatile FIJI plugin for virtual 3D "peeling" of image stacks. The plugin implements spherical and spline surface projections. We applied VolumePeeler to perform peeling in 3D images of spherical embryos, as well as non-spherical tissue layers. The produced images improve the 3D volume visualization and enable analysis and quantification of geometrically challenging microscopy datasets. AVAILABILITY: ImageJ/FIJI software, source code, examples, and tutorials are openly available in https://cimt.uchile.cl/mcerda.

Protocol for extracting live blastoderm cells from embryos of annual killifish.

The implementation of in vitro approaches using undifferentiated embryonic cells from annual killifish to complement existing in vivo developmental studies has been hindered by a lack of efficient isolation techniques. Here, we present a protocol to isolate annual killifish blastoderm cells, at the epiboly and early dispersion phase, from embryos. We describe steps for hair removal, embryo cleaning, dechorionation, and cell purification. This protocol may also be used to develop strategies to isolate cells from embryos presenting similar challenges.

Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) Prepared by Microwave and Ultrasound-Assisted Synthesis: Promising Green Strategies for the Nanoworld.

Many pharmaceutically active molecules are highly lipophilic, which renders their administration and adsorption in patients extremely challenging. Among the countless strategies to overcome this problem, synthetic nanocarriers have demonstrated superb efficiency as drug delivery systems, since encapsulation can effectively prevent a molecules' degradation, thus ensuring increased biodistribution. However, metallic and polymeric nanoparticles have been frequently associated with possible cytotoxic side effects. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), which are prepared with physiologically inert lipids, therefore emerged as an ideal strategy to bypass toxicities issues and avoid the use of organic solvents in their formulations. Different approaches to preparation, using only moderate amounts of external energy to facilitate a homogeneous formation, have been proposed. Greener synthesis strategies have the potential to provide faster reactions, more efficient nucleation, better particle size distribution, lower polydispersities, and furnish products with higher solubility. Particularly microwave-assisted synthesis (MAS) and ultrasound-assisted synthesis (UAS) have been utilized in the manufacturing of nanocarrier systems. This narrative review addresses the chemical aspects of those synthesis strategies and their positive influence on the characteristics of SLNs and NLCs. Furthermore, we discuss the limitations and future challenges for the manufacturing processes of both types of nanoparticles.

A computational framework for testing hypotheses of the minimal mechanical requirements for cell aggregation using early annual killifish embryogenesis as a model.

Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown. Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models. Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations. Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies.

A predictive model of polymetastatic disease from a multicenter large retrospectIve database on colorectal lung metastases treated with stereotactic ablative radiotherapy: The RED LaIT-SABR study.

Aim: Stereotactic ablative radiotherapy (SABR) showed increasing survival in oligometastatic patients. Few studies actually depicted oligometastatic disease (OMD) evolution and which patient will remain disease-free and which will rapidly develop a polymetastatic disease (PMD) after SABR. Therefore, apart from the number of active metastases, there are no clues on which proven factor should be considered for prescribing local treatment in OMD. The study aims to identify predictive factors of polymetastatic evolution in lung oligometastatic colorectal cancer patients. Methods: This international Ethical Committee approved trial (Prot. Negrar 2019-ZT) involved 23 Centers and 450 lung oligometastatic patients. Primary end-point was time to the polymetastatic conversion (tPMC). Additionally, oligometastases number and cumulative gross tumor volume (cumGTV) were used as combined predictive factors of tPMC. Oligometastases number was stratified as 1, 2-3, and 4-5; cumGTV was dichotomized to the value of 10 cc. Results: The median tPMC in the overall population was 26 months. Population was classified in the following tPMC risk classes: low-risk (1-3 oligometastases and cumGTV ≤ 10 cc) with median tPMC of 35.1 months; intermediate-risk (1-3 oligometastases and cumGTV > 10 cc), with median tPMC of 13.9 months, and high-risk (4-5 oligometastases, any cumGTV) with median tPMC of 9.4 months (p = 0.000). Conclusion: The present study identified predictive factors of polymetastatic evolution after SABR in lung oligometastatic colorectal cancer. The results demonstrated that the sole metastases number is not sufficient to define the OMD since patients defined oligometastatic from a numerical point of view might rapidly progress to PMD when the cumulative tumor volume is high. A tailored approach in SABR prescription should be pursued considering the expected disease evolution after SABR, with the aim to avoid unnecessary treatment and toxicity in those at high risk of polymetastatic spread, and maximize local treatment in those with a favorable disease evolution.

Denervation Drives YAP/TAZ Activation in Muscular Fibro/Adipogenic Progenitors.

Loss of motoneuron innervation (denervation) is a hallmark of neurodegeneration and aging of the skeletal muscle. Denervation induces fibrosis, a response attributed to the activation and expansion of resident fibro/adipogenic progenitors (FAPs), i.e., multipotent stromal cells with myofibroblast potential. Using in vivo and in silico approaches, we revealed FAPs as a novel cell population that activates the transcriptional coregulators YAP/TAZ in response to skeletal muscle denervation. Here, we found that denervation induces the expression and transcriptional activity of YAP/TAZ in whole muscle lysates. Using the PdgfraH2B:EGFP/+ transgenic reporter mice to trace FAPs, we demonstrated that denervation leads to increased YAP expression that accumulates within FAPs nuclei. Consistently, re-analysis of published single-nucleus RNA sequencing (snRNA-seq) data indicates that FAPs from denervated muscles have a higher YAP/TAZ signature level than control FAPs. Thus, our work provides the foundations to address the functional role of YAP/TAZ in FAPs in a neurogenic pathological context, which could be applied to develop novel therapeutic approaches for the treatment of muscle disorders triggered by motoneuron degeneration.

Mechanoautophagy: Synergies Between Autophagy and Cell Mechanotransduction at Adhesive Complexes.

Cells are exposed and respond to various mechanical forces and physical cues stemming from their environment. This interaction has been seen to differentially regulate various cellular processes for maintenance of homeostasis, of which autophagy represents one of the major players. In addition, autophagy has been suggested to regulate mechanical functions of the cells including their interaction with the environment. In this minireview, we summarize the state of the art of the fascinating interplay between autophagy and the mechanotransduction machinery associated with cell adhesions, that we name ¨Mechanoautophagy¨.

Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.

Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.

Deep learning for label-free nuclei detection from implicit phase information of mesenchymal stem cells.

Monitoring of adherent cells in culture is routinely performed in biological and clinical laboratories, and it is crucial for large-scale manufacturing of cells needed in cell-based clinical trials and therapies. However, the lack of reliable and easily implementable label-free techniques makes this task laborious and prone to human subjectivity. We present a deep-learning-based processing pipeline that locates and characterizes mesenchymal stem cell nuclei from a few bright-field images captured at various levels of defocus under collimated illumination. Our approach builds upon phase-from-defocus methods in the optics literature and is easily applicable without the need for special microscopy hardware, for example, phase contrast objectives, or explicit phase reconstruction methods that rely on potentially bias-inducing priors. Experiments show that this label-free method can produce accurate cell counts as well as nuclei shape statistics without the need for invasive staining or ultraviolet radiation. We also provide detailed information on how the deep-learning pipeline was designed, built and validated, making it straightforward to adapt our methodology to different types of cells. Finally, we discuss the limitations of our technique and potential future avenues for exploration.

Mechanobiology of Autophagy: The Unexplored Side of Cancer.

Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.

Salvage radiotherapy for oligo-progressive malignant pleural mesothelioma.

OBJECTIVES: No standard treatment option is available for patients with unresectable malignant pleural mesothelioma (MPM) progressing after upfront chemotherapy. We aimed to explore the role of focal radiotherapy (FRT) as a treatment modality for oligo-progressive MPM. MATERIALS AND METHODS: In this retrospective study, consecutive patients pretreated with ≥1 lines of chemotherapy were included. Oligo-progressive MPM was defined as an unresectable disease with radiological progression at ≤3 sites according to a chest-abdominal contrast-enhanced computed tomography. Patients were treated with either stereotactic body radiotherapy (SBRT, ≥5 Gy per fraction) or hypo-fractionated radiotherapy (hypoRT, <5 Gy per fraction). Time to further systemic therapy (TFST) and local control (LC) after FRT were the primary endpoints. Biologically effective dose (BED) was calculated using three different alpha/beta models (1.5 Gy, 3 Gy and 10 Gy). RESULTS: From April 2006 to March 2019, 37 patients were treated on 43 pleural lesions; 16/37 (43 %) had undergone upfront multimodality treatment (MMT) including surgery. FRT was given in 22/37 (59.5 %) after one line of chemotherapy. SBRT was delivered for 26/43 lesions (60.5 %), hypoRT for 17/43 (39.5 %). Median TFST was 6 months (95 % CI 4.9-7.1). LC at 6 months and 1 year was 84 % and 76 %, respectively. Median TFST was longer in patients treated after 1 vs >1 line of chemotherapy (9 vs 4 months, p = 0.001) and in patients pretreated with MMT (6 vs 3 months, p = 0.021). Six-month LC was better in patients treated with a BED > 100 using alpha/beta 1.5 and 3. No ≥ G3 acute or late toxicities were reported. CONCLUSION: FRT was feasible in selected patients with oligo-progressive MPM, allowing delay of further systemic therapies, with no severe toxicity. FRT was more effective when performed at progression after one line of systemic therapy. Our results suggest a radio-resistant behavior of MPM.

Author Correction: Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Two high-yield complementary methods to sort cell populations by their 2D or 3D migration speed.

The potential to migrate is one of the most fundamental functions for various epithelial, mesenchymal, and immune cells. Image analysis of motile cell populations, both primary and cultured, typically reveals an intercellular variability in migration speeds. However, cell migration chromatography, the sorting of large populations of cells based on their migratory characteristics, cannot be easily performed. The lack of such methods has hindered our understanding of the direct correlation between the capacity to migrate and other cellular properties. Here, we report two novel, easily implementable and readily scalable methods to sort millions of live migratory cancer and immune cells based on their spontaneous migration in two-dimensional and three-dimensional microenvironments, respectively. Correlative downstream transcriptomic, molecular and functional tests reveal marked differences between the fast and slow subpopulations in patient-derived cancer cells. We further employ our method to reveal that sorting the most migratory cytotoxic T lymphocytes yields a pool of cells with enhanced cytotoxicity against cancer cells. This phenotypic assay opens new avenues for the precise characterization of the mechanisms underlying hither to unexplained heterogeneities in migratory phenotypes within a cell population, and for the targeted enrichment of the most potent migratory leukocytes in immunotherapies.

Author Correction: Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity.

The Eph family of receptor tyrosine kinases is crucial for assembly and maintenance of healthy tissues. Dysfunction in Eph signaling is causally associated with cancer progression. In breast cancer cells, dysregulated Eph signaling has been linked to alterations in receptor clustering abilities. Here, we implemented a single-cell assay and a scoring scheme to systematically probe the spatial organization of activated EphA receptors in multiple carcinoma cells. We show that cancer cells retain EphA clustering phenotype over several generations, and the degree of clustering reported for migration potential both at population and single-cell levels. Finally, using patient-derived cancer lines, we probed the evolution of EphA signalling in cell populations that underwent metastatic transformation and acquisition of drug resistance. Taken together, our scalable approach provides a reliable scoring scheme for EphA clustering that is consistent over multiple carcinomas and can assay heterogeneity of cancer cell populations in a cost- and time-effective manner.

Functionally Divergent Splicing Variants of the Rice AGAMOUS Ortholog OsMADS3 Are Evolutionary Conserved in Grasses.

Within the MADS-box gene family, the AGAMOUS-subfamily genes are particularly important for plant reproduction, because they control stamen and carpel identity. A number of studies in the last three decades have demonstrated that the AGAMOUS (AG) function has been conserved during land plant evolution. However, gene duplication events have led to subfunctionalization and neofunctionalization of AG-like genes in many species. Here we show that alternative splicing in Oryza sativa produces two variants of the AG ortholog OsMADS3 which differ in just one serine residue, S109. Interestingly, this alternative splicing variant is conserved and specific to the grass family. Since in eudicots the S109 residue is absent in AG proteins, stamen and carpel identity determination activity of the two rice isoforms was tested in Arabidopsis thaliana. These experiments revealed that only the eudicot-like OsMADS3 isoform, lacking the serine residue, had ability to specify stamens and carpels in ag mutant flowers, suggesting an important functional role for the serine residue at position 109 in AG proteins of grasses.