Publications

Cell shape and function are intimately linked, in a way that is mediated by the forces exerted between cells and their environment. The relationship between cell shape and forces has been extensively studied for cells seeded on flat 2D substrates, but not for cells in more physiological 3D settings. Here, a technique called 3D micropatterned traction force microscopy (3D-mu TFM) to confine cells in 3D wells of defined shape, while simultaneously measuring the forces transmitted between cells and their microenvironment is demonstrated. This technique is based on the 3D micropatterning of polyacrylamide wells and on the calculation of 3D traction force from their deformation. With 3D-mu TFM, it is shown that MCF10A breast epithelial cells exert defined, reproducible patterns of forces on their microenvironment, which can be both contractile and extensile. Cells switch from a global contractile to extensile behavior as their volume is reduced are further shown. The technique enables the quantitative study of cell mechanobiology with full access to 3D cellular forces while having accurate control over cell morphology and the mechanical conditions of the microenvironment.

JTD Keywords: Cell volumes, Cytoskeleton, Durotaxis, Micro-wells, Myosi, Reveals, Traction force

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin- proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

JTD Keywords: 3d bioengineered skeletal muscle tissues, Adrenal cortex hormones, Atroph, Colocalization, Corticosteroids, Dexamethasone, Glucocorticoid-receptor, Humans, Mechanisms, Models, biological, Mtor protein, human, Muscle contraction, Muscle fibers, skeletal, Muscle strength, Muscle, skeletal, Muscular diseases, Organ size, Phosphorylation, Proteasome endopeptidase complex, Proto-oncogene proteins c-akt, Receptors, glucocorticoid, Signal transduction, Skeletal-muscle, Steroid myopathy, Steroids, Supplementation, Taurin, Taurine, Tor serine-threonine kinases, Ubiquitin

Surface electromyography (sEMG) can be used to measure the electrical activity of the respiratory muscles. The possible applications of sEMG span from patients suffering from acute respiratory failure to patients receiving chronic home mechanical ventilation, to evaluate muscle function, titrate ventilatory support and guide treatment. However, sEMG is mainly used as a monitoring tool for research and its use in clinical practice is still limited-in part due to a lack of standardization and transparent reporting. During this round table meeting, recommendations on data acquisition, processing, interpretation, and potential clinical applications of respiratory sEMG were discussed. This paper informs the clinical researcher interested in respiratory muscle monitoring about the current state of the art on sEMG, knowledge gaps and potential future applications for patients with respiratory failure.

JTD Keywords: Acute respiratory failure, Artificial ventilation, Asthmatic-children, Breathing muscle, Clinical monitoring, Clinical practice, Clinical research, Consensus development, Data interpretation, Disease exacerbation, Drive, Electrode positioning, Electrode removal, Electromyography, Force, Home care, Human, Human diaphragm, Humans, Information processing, Inspiratory muscle training, Inspiratory muscles, Intensive care unit, Knowledge gap, Long term care, Mechanical ventilation, Medical procedures, Muscle contraction, Muscle fatigue, Muscle function, Muscle training, Muscle, skeletal, Muscle-activity, Noninvasive ventilation, Patient monitoring, Patient-ventilator asynchrony, Physiology, Prognosis, Quality of life, Reporting and data system, Respiratory failure, Respiratory muscles, Review, Severe exacerbations, Signal processing, Skeletal muscle, Standardization, Surface electromyography, Time factor

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

JTD Keywords: 3d cell culture, bacterial cellulose, collagen, composite hydrogels, 3d cell culture, Bacterial cellulose, Cellulose/collagen composite, Collagen, Composite hydrogels, Contraction, Cross-linking, Cytocompatibility, Fibroblasts, Matrix, Mechanical-properties, Reinforcement, Stiffness, Tissue engineering

Abstract Oxidative stress, which occurs when an organism is exposed to an adverse stimulus that results in a misbalance of antioxidant and pro-oxidants species, is the common denominator of diseases considered as a risk factor for SARS-CoV-2 lethality. Indeed, reactive oxygen species caused by oxidative stress have been related to many virus pathogenicity. In this work, simulations have been performed on the receptor binding domain of SARS-CoV-2 spike glycoprotein to study what residues are more susceptible to be attacked by ·OH, which is one of the most reactive radicals associated to oxidative stress. The results indicate that isoleucine (ILE) probably plays a crucial role in modification processes driven by radicals. Accordingly, QM/MM-MD simulations have been conducted to study both the ·OH-mediated hydrogen abstraction of ILE residues and the induced modification of the resulting ILE radical through hydroxylation or nitrosylation reactions. All in all, in silico studies show the importance of the chemical environment triggered by oxidative stress on the modifications of the virus, which is expected to help for foreseeing the identification or development of antioxidants as therapeutic drugs. Graphic abstract

JTD Keywords: atom abstraction, damage, density functionals, hydrogen abstraction, isoleucine, molecular dynamics, pathogenesis, protein, reactive oxygen species, receptor binding domain, residues, spike protein, Amino-acids, Hydrogen abstraction, Isoleucine, Molecular dynamics, Reactive oxygen species, Receptor binding domain, Spike protein

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin β1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.

JTD Keywords: actin, activation, actomyosin, adherens junctions, adhesion, e-cadherin, egf, maturation, mechanical regulation, micropatterning, migration, traction forces, transduction, transmission, Actomyosin, Adherens junctions, Cell adhesion, Cell membrane, Collective contractility, Egf, Epidermal growth factor, Epidermal-growth-factor, Epithelial cells, Micropatterning, Myosins, Traction forces

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration.Copyright © 2022 Elsevier Inc. All rights reserved.

JTD Keywords: activation, c-met, contact inhibition, focal adhesions, heparan-sulfate, mechanical forces, morphogenesis, rho, stress fibers, Collective cell migration, Erk, Feedback regulation, Fret, Growth-factor receptor, Hgf, Lamellipodia, Leader cell specification, Signal transduction, Traction force, Wound healing

In recent years, 3D in vitro modeling of human skeletal muscle has emerged as a subject of increasing interest, due to its applicability in basic studies or screening platforms. These models strive to recapitulate key features of muscle architecture and function, such as cell alignment, maturation, and contractility in response to different stimuli. To this end, it is required to culture cells in biomimetic hydrogels suspended between two anchors. Currently available protocols are often complex to produce, have a high rate of breakage, or are not adapted to imaging and stimulation. Therefore, we sought to develop a simplified and reliable protocol, which still enabled versatility in the study of muscle function. In our method, we have used human immortalized myoblasts cultured in a hydrogel composed of MatrigelTM and fibrinogen, to create muscle strips suspended between two VELCROTM anchors. The resulting muscle constructs show a differentiated phenotype and contractile activity in response to electrical, chemical and optical stimulation. This activity is analyzed by two alternative methods, namely contraction analysis and calcium analysis with Fluo-4 AM. In all, our protocol provides an optimized version of previously published methods, enabling individual imaging of muscle bundles and straightforward analysis of muscle response with standard image analysis software. This system provides a start-to-finish guide on how to produce, validate, stimulate, and analyze bioengineered muscle. This ensures that the system can be quickly established by researchers with varying degrees of expertise, while maintaining reliability and similarity to native muscle.

JTD Keywords: cells, contraction, models, Differentiation

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

JTD Keywords: atomic force microscopy, cajal-retzius cells, cortical development, marginal zone, mechanical cues, Atomic force microscopy, Cajal-retzius cells, Central-nervous-system, Cortical development, Cortical hem, Developing cerebral-cortex, Expression, Growth, Marginal zone, Mechanical cues, Mouse, Neuronal migration, Nogo receptor, Olfactory ensheathing cells, Tangential migration, Traction force microscopy

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

JTD Keywords: actomyosin, central nervous system, drosophila, glia, mechanics, morphogenesis, neuron, ventral nerve cord, Actomyosin, Animals, Central nervous system, Collagen-iv, Contraction, Drosophila, Embryonic development, Forces, Gene, Glia, Glial-cells, Mechanics, Migration, Morphogenesis, Neuroglia, Neuron, Neurons, Quantification, System, Tissue, Ventral nerve cord, Viscolelastic model

Research into the development of therapeutic strategies is basedmainly on animal models and cell cultures. The ability to extrapolatedata from them is limited, and research on new drugs cannot beperformed efficiently. This is especially dramatic in rare diseases,which are intrinsically very heterogeneous. The generation of ad-vanced models using tissue engineering and patient-derived cellsallows fabricating new platforms for studying pathological processesand discovering new potential drugs. Here, we developed a patient-derived 3D functional skeletal muscle for Duchenne muscular dys-trophy (DMD). DMD is the most prevalent neuromuscular diseasediagnosed during childhood. The disease is characterized by pro-gressive degeneration of skeletal and cardiac muscle caused by thelack of dystrophin protein. Although there are several molecules indrug development for DMD, there is no treatment available for pa-tients to date. By using a 3D-printed casting mold, we encapsulatedpatient-derived myogenic precursor cells in a fibrin-composite ma-trix. This platform incorporated two flexible T-shaped pillars thatprovided continuous tension to the tissue, thus allowing the orien-tation of the muscle fibers. Our 3D muscle model expressed maturemuscle markers and responded to electric pulse stimulation (EPS).Besides, contraction dynamics between DMD and control tissueswere shown to be different. Moreover, an increase of damagemarkers after EPS was observed in DMD but not in healthy tissues.Finally, the tissues will be integrated into a microfluidic device tomonitor drug administration. Eventually, the microfluidic systemwill be connected to a biosensors system for the real-time detectionof biomarkers.

JTD Keywords: Casting, Contraction dynamics, Muscular dystrophy

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

The dizzying life of the homeostatic intestinal epithelium is governed by a complex interplay between fate, form, force and function. This interplay is beginning to be elucidated thanks to advances in intravital and ex vivo imaging, organoid culture, and biomechanical measurements. Recent discoveries have untangled the intricate organization of the forces that fold the monolayer into crypts and villi, compartmentalize cell types, direct cell migration, and regulate cell identity, proliferation and death. These findings revealed that the dynamic equilibrium of the healthy intestinal epithelium relies on its ability to precisely coordinate tractions and tensions in space and time. In this review, we discuss recent findings in intestinal mechanobiology, and highlight some of the many fascinating questions that remain to be addressed in this emerging field.Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.

JTD Keywords: crypt fission, designer matrices, differentiation, growth, gut, migration, model, scaffold, tissue mechanics, Biophysics, Cell migration, Cell movement, Cell proliferation, Ex vivo study, Human tissue, Intestinal mucosa, Intestine epithelium, Monolayer culture, Organoid, Organoids, Review, Stem-cell, Tension, Traction therapy

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

JTD Keywords: cell wall, efficacy, glycerol, hydrophobicity, lipid, neutral red, pdim, pgl, protein, strains, viability, virulence, Acylglycerol, Albumin, Article, Asparagine, Bacterial cell wall, Bacterial gene, Bacterium culture, Bcg vaccine, Catalase, Cell wall, Chloroform, Controlled study, Escherichia coli, Gene expression, Genomic dna, Glycerol, Glycerol monomycolate, Hexadecane, Housekeeping gene, Hydrophobicity, Immune response, Immunogenicity, Immunotherapy, Lipid, Lipid fingerprinting, Magnesium sulfate, Mercaptoethanol, Methanol, Methylglyoxal, Molybdatophosphoric acid, Mycobacterium bovis bcg, Neutral red, Nonhuman, Pdim, Petroleum ether, Pgl, Phenotype, Physical chemistry, Real time reverse transcription polymerase chain reaction, Rna 16s, Rna extraction, Rv0577, Staining, Thin layer chromatography, Unclassified drug

[Figure: see text].

JTD Keywords: activation, cell extrusion, contraction, drives, homeostasis, interface, junctions, kinase, tension, Adhesion, Article, Cell membranes, Chemical activation, Cytology, E-cadherin, Epithelial monolayers, Epithelium, Homoeostasis, Human, Mechanical instabilities, Monolayers, Morphological transformations, Morphology, Normal tissue, Oncogenics, Soft substrates, Substrates, Tissue, Tissue mechanics, Tissue morphology, Tumor development

Gas chromatography—ion mobility spectrometry (GC-IMS) allows the fast, reliable, and inexpensive chemical composition analysis of volatile mixtures. This sensing technology has been successfully employed in food science to determine food origin, freshness and preventing alimentary fraud. However, GC-IMS data is highly dimensional, complex, and suffers from strong non-linearities, baseline problems, misalignments, peak overlaps, long peak tails, etc., all of which must be corrected to properly extract the relevant features from samples. In this work, a pipeline for signal pre-processing, followed by four different approaches for feature extraction in GC-IMS data, is presented. More precisely, these approaches consist of extracting data features from: (1) the total area of the reactant ion peak chromatogram (RIC); (2) the full RIC response; (3) the unfolded sample matrix; and (4) the ion peak volumes. The resulting pipelines for data processing were applied to a dataset consisting of two different quality class Iberian ham samples, based on their feeding regime. The ability to infer chemical information from samples was tested by comparing the classification results obtained from partial least-squares discriminant analysis (PLS-DA) and the samples’ variable importance for projection (VIP) scores. The choice of a feature extraction strategy is a trade-off between the amount of chemical information that is preserved, and the computational effort required to generate the data models.

JTD Keywords: authenticity, classification, electronic-nose, feature extraction, food analysis, gc-ims, headspace, least-squares, models, pld-da, pre-processing, quality, sensory analysis, wine, Feature extraction, Food analysis, Gc-ims, Hs-gc-ims, Pld-da, Pre-processing

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

Bioinspired hybrid soft robots that combine living and synthetic components are an emerging field in the development of advanced actuators and other robotic platforms (i.e., swimmers, crawlers, and walkers). The integration of biological components offers unique characteristics that artificial materials cannot precisely replicate, such as adaptability and response to external stimuli. Here, we present a skeletal muscle–based swimming biobot with a three-dimensional (3D)–printed serpentine spring skeleton that provides mechanical integrity and self-stimulation during the cell maturation process. The restoring force inherent to the spring system allows a dynamic skeleton compliance upon spontaneous muscle contraction, leading to a cyclic mechanical stimulation process that improves the muscle force output without external stimuli. Optimization of the 3D-printed skeletons is carried out by studying the geometrical stiffnesses of different designs via finite element analysis. Upon electrical actuation of the muscle tissue, two types of motion mechanisms are experimentally observed: directional swimming when the biobot is at the liquid-air interface and coasting motion when it is near the bottom surface. The integrated compliant skeleton provides both the mechanical self-stimulation and the required asymmetry for directional motion, displaying its maximum velocity at 5 hertz (800 micrometers per second, 3 body lengths per second). This skeletal muscle–based biohybrid swimmer attains speeds comparable with those of cardiac-based biohybrid robots and outperforms other muscle-based swimmers. The integration of serpentine-like structures in hybrid robotic systems allows self-stimulation processes that could lead to higher force outputs in current and future biomimetic robotic platforms. Copyright © 2021 The Authors, some rights reserved;

JTD Keywords: actuators, design, fabrication, mechanics, mems, myotubes, platform, tissue, 3d printers, Agricultural robots, Biological components, Biomimetic processes, Electrical actuation, Geometrical stiffness, Intelligent robots, Liquefied gases, Liquid-air interface, Mechanical integrity, Mechanical stimulation, Muscle, Muscle contractions, Phase interfaces, Robotics, Serpentine, Springs (components), Threedimensional (3-d)

This paper describes the design of a linear phase low-pass differentiator filter with a finite impulse response (FIR) for extracting transient features of gas sensor signals (the so-called “bouts”). The detection of these bouts is relevant for estimating the distance of a gas source in a turbulent plume. Our current proposal addresses the shortcomings of previous ‘bout’ estimation methods, namely: (i) they were based in non-causal digital filters precluding real time operation, (ii) they used non-linear phase filters leading to waveform distortions and (iii) the smoothing action was achieved by two filters in cascade, precluding an easy tuning of filter performance. The presented method is based on a low-pass FIR differentiator, plus proper post-processing, allowing easy algorithmic implementation for real-time robotic exploration. Linear phase filters preserve signal waveform in the bandpass region for maximum reliability concerning both ‘bout’ detection and amplitude estimation. As a case study, we apply the proposed filter to predict the source distance from recordings obtained with metal oxide (MOX) gas sensors in a wind tunnel. We first perform a joint optimization of the cut-off frequency of the filter and the bout amplitude threshold, for different wind speeds, uncovering interesting relationships between these two parameters. We demonstrate that certain combinations of parameters can reduce the prediction error to 8 cm (in a distance range of 1.45 m) improving previously reported performances in the same dataset by a factor of 2.5. These results are benchmarked against traditional source distance estimators such as the mean, variance and maximum of the response. We also study how the length of the measurement window affects the performance of different signal features, and how to select the filter parameters to make the predictive models more robust to changes in wind speed. Finally, we provide a MATLAB implementation of the bout detection algorithm and all analysis code used in this study.

JTD Keywords: Gas sensors, Differentiator, Low pass filter, Metal oxide semiconductor, MOX sensors, Signal processing, Feature extraction, Gas source localization, Robotics

Galleria mellonella larvae are an alternative in vivo model that has been extensively used to study the virulence and pathogenicity of different bacteria due to its practicality and lack of ethical constraints. However, the larvae possess intrinsic autofluorescence that obstructs the use of fluorescent proteins to study bacterial infections, hence better methodologies are needed. Here, we report the construction of a promoter probe vector with bioluminescence expression as well as the optimization of a total bacterial RNA extraction protocol to enhance the monitoring of in vivo infections. By employing the vector to construct different gene promoter fusions, variable gene expression levels were efficiently measured in G. mellonella larvae at various time points during the course of infection and without much manipulation of the larvae. Additionally, our optimized RNA extraction protocol facilitates the study of transcriptional gene levels during an in vivo infection. The proposed methodologies will greatly benefit bacterial infection studies as they can contribute to a better understanding of the in vivo infection processes and pathogen–mammalian host interactions.

JTD Keywords: Galleria mellonella, P. aeruginosa, Hemolymph, Hemocytes, Bioluminescence, Promoter probe vector, Optimized RNA extraction, Ribonucleotide reductases

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

JTD Keywords: 3-D hydrogel cultures, Axonal growth, Cell transfection, Chemoattraction, Chemorepulsion, Embryonic nervous system, Issue 148, Neuroscience, Tissue explants

This paper explores which signal features of a gas sensor are optimum for assessing the proximity to a gas source in an open environment. Specifically, we compare three statistical descriptors of the signal (mean, variance and maximum response) against the 'bout' frequency, a feature computed in the derivative of the response. The experimental setup includes a generator of turbulent plumes and a sensing board composed of three metal oxide (MOX) sensors of different types. The main conclusion is that the maximum response is the most robust feature across the three sensors. The 'bout' frequency can be very sensitive to an additional parameter (the noise threshold).

JTD Keywords: Feature extraction, Gas plume, Gas sensors, Gas source localization, MOX, Signal processing

Estimating the affective state of a user during a computer task traditionally relies on either subjective reports or analysis of physiological signals, facial expressions, and other measures. These methods have known limitations, can be intrusive and may require specialized equipment. An alternative would be employing a ubiquitous device of everyday use such as a standard keyboard. Here we investigate if we can infer the emotional state of a user by analyzing their typing patterns. To test this hypothesis, we asked 400 participants to caption a set of emotionally charged images taken from a standard database with known ratings of arousal and valence. We computed different keystroke pattern dynamics, including keystroke duration (dwell time) and latency (flight time). By computing the mean value of all of these features for each image, we found a statistically significant negative correlation between dwell times and valence, and between flight times and arousal. These results highlight the potential of using keystroke dynamics to estimate the affective state of a user in a non-obtrusive way and without the need for specialized devices.

JTD Keywords: Feature extraction, Correlation, Keyboards, Task analysis, Statistical analysis, Affective computing, Standards, Keystroke, Keyboard, Typing, Arousal, Valence, Affect

The components of a Living Machine must be integrated into a functioning whole, which requires a detailed understanding of the architecture of living machines. This chapter starts with a conceptual and historical analysis which from Plato brings us to nineteenth-century neuroscience and early concepts of the layered structure of nervous systems. These concepts were further captured in the cognitive behaviorism of Tolman and came to full fruition in the cognitive revolution of the second half of the twentieth century. Verschure subsequently describes the most relevant proposals of cognitive architectures followed by an overview of the few proposals stemming from modern neuroscience on the architecture of the brain. Subsequently, we will look at contemporary contenders that mediate between cognitive and brain architecture. An important challenge to any model of cognitive architectures is how to benchmark it. Verschure proposes the Unified Theories of Embodied Minds (UTEM) benchmark which advances from Newell’s classic Unified Theories of Cognition benchmark.

JTD Keywords: Architecture, Mind, Brain, Organization, System, Virtualization, Abstraction layers

Portal hypertension either develops due to progressive liver fibrosis or is the consequence of vascular liver diseases such as portal vein thrombosis or non-cirrhotic portal hypertension. This chapter focuses on different rodent models of liver fibrosis with portal hypertension and also in few non-cirrhotic portal hypertension models. Importantly, after the development of portal hypertension, the proper assessment of drug effects in the portal and systemic circulation should be discussed. The last part of the chapter is dedicated in these techniques to assess the in vivo hemodynamics and the ex vivo techniques of the isolated liver perfusion and vascular contractility.

JTD Keywords: Aortic ring contraction, Bile duct ligation, Carbon tetrachloride, Colored microsphere technique, High-fat diet, Isolated in situ liver perfusion, Methionine-choline-deficient diet, Partial portal vein ligation, Portal hypertension

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization.

JTD Keywords: Epiblast, Human embryonic stem cells, Mechanotransduction, Monolayer stress microscopy, Self-organization, Traction force

Differentiating normal from adventitious respiratory sounds (RS) is a major challenge in the diagnosis of pulmonary diseases. Particularly, continuous adventitious sounds (CAS) are of clinical interest because they reflect the severity of certain diseases. This study presents a new classifier that automatically distinguishes normal sounds from CAS. It is based on the multi-scale analysis of instantaneous frequency (IF) and envelope (IE) calculated after ensemble empirical mode decomposition (EEMD). These techniques have two major advantages over previous techniques: high temporal resolution is achieved by calculating IF-IE and a priori knowledge of signal characteristics is not required for EEMD. The classifier is based on the fact that the IF dispersion of RS signals markedly decreases when CAS appear in respiratory cycles. Therefore, CAS were detected by using a moving window to calculate the dispersion of IF sequences. The study dataset contained 1494 RS segments extracted from 870 inspiratory cycles recorded from 30 patients with asthma. All cycles and their RS segments were previously classified as containing normal sounds or CAS by a highly experienced physician to obtain a gold standard classification. A support vector machine classifier was trained and tested using an iterative procedure in which the dataset was randomly divided into training (65%) and testing (35%) sets inside a loop. The SVM classifier was also tested on 4592 simulated CAS cycles. High total accuracy was obtained with both recorded (94.6% ± 0.3%) and simulated (92.8% ± 3.6%) signals. We conclude that the proposed method is promising for RS analysis and classification.

JTD Keywords: Diseases, Dispersion, Empirical mode decomposition, Feature extraction, Informatics, Support vector machines

Changes in the left ventricle function produce alternans in the hemodynamic and electric behavior of the cardiovascular system. A total of 49 cardiomyopathy patients have been studied based on the blood pressure signal (BP), and were classified according to the left ventricular ejection fraction (LVEF) in low risk (LR: LVEF>35%, 17 patients) and high risk (HR: LVEF≤35, 32 patients) groups. We propose to characterize these patients using a linear and a nonlinear methods, based on the spectral estimation and the recurrence plot, respectively. From BP signal, we extracted each systolic time interval (STI), upward systolic slope (BPsl), and the difference between systolic and diastolic BP, defined as pulse pressure (PP). After, the best subset of parameters were obtained through the sequential feature selection (SFS) method. According to the results, the best classification was obtained using a combination of linear and nonlinear features from STI and PP parameters. For STI, the best combination was obtained considering the frequency peak and the diagonal structures of RP, with an area under the curve (AUC) of 79%. The same results were obtained when comparing PP values. Consequently, the use of combined linear and nonlinear parameters could improve the risk stratification of cardiomyopathy patients.

JTD Keywords: Feature extraction, Blood pressure, Heart rate, Estimation, Data mining, Covariance matrices, Hospitals

Aging population is a major concern that is reflected in the increase of chronic diseases. Heart Failure (HF) is one of the most common chronic diseases of elderly people that is punctuated with acute episodes, which result in hospitalization. The periodic modulation of the amplitude of the breathing pattern is proved to be one of the multiple symptoms of an acute episode, and thus, the features extracted from its characterization contribute in the improvement of the first diagnosis of the clinical practice. The main objective of this study is to evaluate if the features extracted from the breathing pattern along with common clinical variables are reliable enough to detect Periodic Breathing (PB). A dataset of 44 elderly patients containing clinical information and a short record of electrocardiogram and respiratory flow signal was used to train two machine learning classification methods: Support Vector Machine (SVM) and Linear Discriminant Analysis (LDA). All the available clinical parameters within the dataset along with the parameters characterizing the respiratory pattern were used to classify the observations into two groups. SVM classification was optimized and performed using a = -8 and C = 10.04 giving an accuracy of 88.2 % sensitivity of 90 % and specificity of 85.7 % Similar results were achieved with LDA classifying with an accuracy of 82.4 %, a sensitivity of 81.8% and specificity of 83.3 % PB has been accurately detected using both classifiers.

JTD Keywords: Support vector machines, Feature extraction, Training, Senior citizens, Standards, Training data, Hospitals

Adherent cells exert traction forces on their substrate, and these forces play important roles in biological functions such as mechanosensing, cell differentiation and cancer invasion. The method of choice to assess these active forces is traction force microscopy (TFM). Despite recent advances, TFM remains highly sensitive to measurement noise and exhibits limited spatial resolution. To improve the resolution and noise robustness of TFM, here we adapt techniques from compressed sensing (CS) to the reconstruction of the traction field from the substrate displacement field. CS enables the recovery of sparse signals at higher resolution from lower resolution data. Focal adhesions (FAs) of adherent cells are spatially sparse implying that traction fields are also sparse. Here we show, by simulation and by experiment, that the CS approach enables circumventing the Nyquist-Shannon sampling theorem to faithfully reconstruct the traction field at a higher resolution than that of the displacement field. This allows reaching state-of-the-art resolution using only a medium magnification objective. We also find that CS improves reconstruction quality in the presence of noise. Statement of Significance A great scientific advance of the past decade is the recognition that physical forces determine an increasing list of biological processes. Traction force microscopy which measures the forces that cells exert on their surroundings has seen significant recent improvements, however the technique remains sensitive to measurement noise and severely limited in spatial resolution. We exploit the fact that the force fields are sparse to boost the spatial resolution and noise robustness by applying ideas from compressed sensing. The novel method allows high resolution on a larger field of view. This may in turn allow better understanding of the cell forces at the multicellular level, which are known to be important in wound healing and cancer invasion.

JTD Keywords: Compressed sensing, High resolution, Traction force microscopy

Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion.

JTD Keywords: Olfactory ensheathing cells, Traction force microscopy, Chondroitin sulphate proteoglycans, Cell migration, Nogo receptor ectodomain

Calcium signaling participates in different cellular processes leading to cell migration. TRPV4, a non-selective cation channel that responds to mechano-osmotic stimulation and heat, is also involved in cell migration. However, the mechanistic involvement of TRPV4 in cell migration is currently unknown. We now report that expression of the mutant channel TRPV4-121AAWAA (lacking the phosphoinositide-binding site 121KRWRK125 and the response to physiological stimuli) altered HEK293 cell migration. Altered migration patterns included periods of fast and persistent motion followed by periods of stalling and turning, and the extension of multiple long cellular protrusions. TRPV4-WT overexpressing cells showed almost complete loss of directionality with frequent turns, no progression, and absence of long protrusions. Traction microscopy revealed higher tractions forces in the tail of TRPV4-121AAWAA than in TRPV4-WT expressing cells. These results are consistent with a defective and augmented tail retraction in TRPV4-121AAWAA- and TRPV4-WT-expressing cells, respectively. The activity of calpain, a protease implicated in focal adhesion (FA) disassembly, was decreased in TRPV4-121AAWAA compared with TRPV4-WT-expressing cells. Consistently, larger focal adhesions were seen in TRPV4-121AAWAA compared with TRPV4-WT-expressing HEK293 cells, a result that was also reproduced in T47D and U87 cells. Similarly, overexpression of the pore-dead mutant TRPV4-M680D resumed the TRPV4-121AAWAA phenotype presenting larger FA. The migratory phenotype obtained in HEK293 cells overexpressing TRPV4-121AAWAA was mimicked by knocking-down TRPC1, a cationic channel that participates in cell migration. Together, our results point to the participation of TRPV4 in the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels or proteins present at the FA sites.

JTD Keywords: Calcium, Calpain, Focal adhesion, Migration, Traction forces, TRPV4

Abstract Fundamental biological processes including morphogenesis and tissue repair require cells to migrate collectively. In these processes, epithelial or endothelial cells move in a cooperative manner coupled by intercellular junctions. Ultimately, the movement of these multicellular systems occurs through the generation of cellular forces, exerted either on the substrate via focal adhesions (cell–substrate forces) or on neighboring cells through cell–cell junctions (cell–cell forces). Quantitative measurements of multicellular forces and kinematics with cellular or subcellular resolution have become possible only in recent years. In this chapter, we describe some of these techniques, which include particle image velocimetry to map cell velocities, traction force microscopy to map forces exerted by cells on the substrate, and monolayer stress microscopy to map forces within and between cells. We also describe experimental protocols to perform these measurements. The combination of these techniques with high-resolution imaging tools and molecular perturbations will lead to a better understanding of the mechanisms underlying collective cell migration in health and disease.

JTD Keywords: Collective cell migration, Monolayer stress microscopy, Traction force microscopy

We have evaluated the behavior of single-walled carbon nanohorns as a sorbent for headspace and direct immersion (micro)solid phase extraction using volatile organic compounds (VOCs) as model analytes. The conical carbon nanohorns were first oxidized in order to increase their solubility in water and organic solvents. A microporous hollow polypropylene fiber served as a mechanical support that provides a high surface area for nanoparticle retention. The extraction unit was directly placed in the liquid sample or the headspace of an aqueous standard or a water sample to extract and preconcentrate the VOCs. The variables affecting extraction have been optimized. The VOCs were then identified and quantified by GC/MS. We conclude that direct immersion of the fiber is the most adequate method for the extraction of VOCs from both liquid samples and headspace. Detection limits range from 3.5 to 4.3 ng L-1 (excepted for toluene with 25 ng L-1), and the precision (expressed as relative standard deviation) is between 3.9 and 9.6 %. The method was applied to the determination of toluene, ethylbenzene, various xylene isomers and styrene in bottled, river and tap waters, and the respective average recoveries of spiked samples are 95.6, 98.2 and 86.0 %.

JTD Keywords: (Micro)solid phase extraction, Nanotechnology, Oxidized single-walled carbon nanohorns, Volatiles compounds, Waters

Ischemic and dilated cardiomyopathy are associated with disorders of myocardium. Using the blood pressure (BP) signal and the values of the ventricular ejection fraction, we obtained parameters for stratifying cardiomyopathy patients as low- and high-risk. We studied 48 cardiomyopathy patients characterized by NYHA ≥2: 19 patients with dilated cardiomyopathy (DCM) and 29 patients with ischemic cardiomyopathy (ICM). The left ventricular ejection fraction (LVEF) percentage was used to classify patients in low risk (LR: LVEF > 35%, 17 patients) and high risk (HR: LVEF ≤ 35%, 31 patients) groups. From the BP signal, we extracted the upward systolic slope (BP_sl), the difference between systolic and diastolic BP (BPA), and systolic time intervals (STI). When we compared the LR and HR groups in the time domain analysis, the best parameters were standard deviation (SD) of 1=STI, kurtosis (K) of BP_sl, and K of BPA. In the frequency domain analysis, very low frequency (VLF) and high frequency (HF) bands showed statistically significant differences in comaprisons of LR and HR groups. The area under the curve of power spectral density was the best parameter in all classifications, and particularly in the very-low-and high- frequency bands (p <; 0.001). These parameters could help to improve the risk stratification of cardiomyopathy patients.

JTD Keywords: blood pressure measurement, cardiovascular system, diseases, medical disorders, medical signal processing, statistical analysis, time-domain analysis, BP signal, HR groups, LR groups, blood pressure signal analysis, cardiomyopathy patients, diastolic BP, dilated cardiomyopathy, frequency domain analysis, high-frequency bands, ischemic cardiomyopathy, left ventricular ejection fraction, low-frequency bands, myocardium disorders, patient characterization, power spectral density curve, standard deviation, statistical significant differences, systolic BP, systolic slope, systolic time intervals, time domain analysis, ventricular ejection fraction, Abstracts, Databases, Parameter extraction, Telecommunication standards, Time-frequency analysis

One of the most challenging problems in intensive care is still the process of discontinuing mechanical ventilation, called weaning process. Both an unnecessary delay in the discontinuation process and a weaning trial that is undertaken too early are undesirable. In this study, we analyzed respiratory pattern variability using the respiratory volume signal of patients submitted to two different levels of pressure support ventilation (PSV), prior to withdrawal of the mechanical ventilation. In order to characterize the respiratory pattern, we analyzed the following time series: inspiratory time, expiratory time, breath duration, tidal volume, fractional inspiratory time, mean inspiratory flow and rapid shallow breathing. Several autoregressive modeling techniques were considered: autoregressive models (AR), autoregressive moving average models (ARMA), and autoregressive models with exogenous input (ARX). The following classification methods were used: logistic regression (LR), linear discriminant analysis (LDA) and support vector machines (SVM). 20 patients on weaning trials from mechanical ventilation were analyzed. The patients, submitted to two different levels of PSV, were classified as low PSV and high PSV. The variability of the respiratory patterns of these patients were analyzed. The most relevant parameters were extracted using the classifiers methods. The best results were obtained with the interquartile range and the final prediction errors of AR, ARMA and ARX models. An accuracy of 95% (93% sensitivity and 90% specificity) was obtained when the interquartile range of the expiratory time and the breath duration time series were used a LDA model. All classifiers showed a good compromise between sensitivity and specificity.

JTD Keywords: autoregressive moving average processes, feature extraction, medical signal processing, patient care, pneumodynamics, signal classification, support vector machines, time series, ARX, autoregressive modeling techniques, autoregressive models with exogenous input, autoregressive moving average model, breath duration time series, classification method, classifier method, discontinuing mechanical ventilation, expiratory time, feature extraction, final prediction errors, fractional inspiratory time, intensive care, interquartile range, linear discriminant analysis, logistic regression analysis, mean inspiratory flow, patient respiratory volume signal, pressure support level, pressure support ventilation, rapid shallow breathing, respiratory pattern variability characterization, support vector machines, tidal volume, weaning trial, Analytical models, Autoregressive processes, Biological system modeling, Estimation, Support vector machines, Time series analysis, Ventilation

This work investigates the very low frequency (VLF) modulation of QRS slopes and heart rate variability (HRV). Electrocardiogram (ECG) and respiratory flow signal were acquired from patients with dilated cardiomyopathy and ischemic cardiomyopathy. HRV as well as the upward QRS slope (I_US) and downward QRS slope (I_DS) were extracted from the ECG. The relation between HRV and QRS slopes in the VLF band was measured using ordinary coherence in 5-minute segments. Partial coherence was then used to remove the influence that respiration simultaneously exerts on HRV and QRS slopes. A statistical threshold was determined, below which coherence values were considered not to represent a linear relation. 7 out of 276 segments belonging to 5 out of 29 patients for I_US and 10 segments belonging to 5 patients for I_DS presented a VLF modulation in QRS slopes, HRV and respiration. In these segments spectral coherence was statistically significant, while partial coherence decreased, indicating that the coupling HRV and QRS slopes was related to respiration. 4 segments had a partial coherence value below the threshold for I_US, 3 segments for I_DS. The rest of the segments also presented a notable decrease in partial coherence, but still above the threshold, which means that other non-linearly effects may also affect this modulation.

JTD Keywords: diseases, electrocardiography, feature extraction, medical signal processing, pneumodynamics, statistical analysis, ECG, QRS slopes, cardiomyopathy patients, dilated cardiomyopathy, electrocardiogram, feature extraction, heart rate variability, ischemic cardiomyopathy, ordinary coherence, partial coherence value, respiration, respiratory flow signal acquisition, spectral coherence, statistical threshold, time 5 min, very low frequency modulation, Coherence, Educational institutions, Electrocardiography, Frequency modulation, Heart rate variability

Some of the most common clinical problems in elderly patients are related to diseases of the cardiac and respiratory systems. Elderly patients often have altered breathing patterns, such as periodic breathing (PB) and Cheyne-Stokes respiration (CSR), which may coincide with chronic heart failure. In this study, we used the envelope of the respiratory flow signal to characterize respiratory patterns in elderly patients. To study different breathing patterns in the same patient, the signals were segmented into windows of 5 min. In oscillatory breathing patterns, frequency and time-frequency parameters that characterize the discriminant band were evaluated to identify periodic and non-periodic breathing (PB and nPB). In order to evaluate the accuracy of this characterization, we used a feature selection process, followed by linear discriminant analysis. 22 elderly patients (7 patients with PB and 15 with nPB pattern) were studied. The following classification problems were analyzed: patients with either PB (with and without apnea) or nPB patterns, and patients with CSR versus PB, CSR versus nPB and PB versus nPB patterns. The results showed 81.8% accuracy in the comparisons of nPB and PB patients, using the power of the modulation peak. For the segmented signal, the power of the modulation peak, the frequency variability and the interquartile ranges provided the best results with 84.8% accuracy, for classifying nPB and PB patients.

JTD Keywords: cardiovascular system, diseases, feature extraction, geriatrics, medical signal processing, oscillations, pneumodynamics, signal classification, time-frequency analysis, Cheyne-Stokes respiration, apnea, cardiac systems, chronic heart failure, classification problems, discriminant band, diseases, elderly patients, feature selection process, frequency variability, interquartile ranges, linear discriminant analysis, nonperiodic breathing, oscillatory breathing pattern, periodic breathing, respiratory How signal, respiratory systems, signal segmentation, time 5 min, time-frequency parameters, Accuracy, Aging, Frequency modulation, Heart, Senior citizens, Time-frequency analysis

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell–cell and cell–matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40.

JTD Keywords: Ensheathing glia, Traction force, microscopy, Migration, Myelin-associated inhibitors

A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer.

JTD Keywords: Contraction, Human umbilical vein endothelial cells, Permeability, Traction force, Cell-cell contact, Cell-substrate contact, Substrate stiffness, Rho kinase, Vascular endothelial cadherin, Thrombin

Matrix and tissue rigidity guides many cellular processes, including the differentiation of stem cells and the migration of cells in health and disease. Cells actively and transiently test rigidity using mechanisms limited by inherent physical parameters that include the strength of extracellular attachments, the pulling capacity on these attachments, and the sensitivity of the mechanotransduction system. Here, we focus on rigidity sensing mediated through the integrin family of extracellular matrix receptors and linked proteins and discuss the evidence supporting these proteins as mechanosensors.

JTD Keywords: Focal adhesion kinase, Atomic Force Microscopy, Smooth-muscle cells, Traction forces, Living cells, Mechanical force, Locomoting cells

The differentiation of obstructive and central respiratory events is a major challenge in the diagnosis of sleep disordered breathing. Esophageal pressure (Pes) measurement is the gold-standard method to identify these events but its invasiveness deters its usage in clinical routine. Flattening patterns appear in the airflow signal during episodes of inspiratory flow limitation (IFL) and have been shown with invasive techniques to be useful to differentiate between central and obstructive hypopneas. In this study we present a new method for the automatic non-invasive differentiation of obstructive and central hypopneas solely with nasal airflow. An overall of 36 patients underwent full night polysomnography with systematic Pes recording and a total of 1069 hypopneas were manually scored by human experts to create a gold-standard annotation set. Features were automatically extracted from the nasal airflow signal to train and test our automatic classifier (Discriminant Analysis). Flattening patterns were non-invasively assessed in the airflow signal using spectral and time analysis. The automatic non-invasive classifier obtained a sensitivity of 0.71 and an accuracy of 0.69, similar to the results obtained with a manual non-invasive classification algorithm. Hence, flattening airflow patterns seem promising for the non-invasive differentiation of obstructive and central hypopneas.

JTD Keywords: Practical, Experimental/ biomedical measurement, Feature extraction, Flow measurement, Medical disorders, Medical signal processing, Patient diagnosis, Pneumodynamics, Pressure measurement, Signal classification, Sleep, Spectral analysis/ automatic noninvasive differentiation, Obstructive hypopnea, Central hypopnea, Inspiratory flow limitation, Nasal airflow, Esophageal pressure, Polysomnography, Feature extraction, Discriminant analysis, Spectral analysis

Sleep Apnea-Hypopnea Syndrome (SAHS) diagnosis is still done with an overnight multi-channel polysomnography. Several efforts are being made to study profoundly the snore mechanism and discover how it can provide an opportunity to diagnose the disease. This work introduces the concept of regular snores, defined as the ones produced in consecutive respiratory cycles, since they are produced in a regular way, without interruptions. We applied 2 thresholds (TH/sub adaptive/ and TH/sub median/) to the time interval between successive snores of 34 subjects in order to select regular snores from the whole all-night snore sequence. Afterwards, we studied the effectiveness that parameters, such as time interval between successive snores and the mean intensity of snores, have on distinguishing between different levels of SAHS severity (AHI (Apnea-Hypopnea Index)<5h/sup -1/, AHI<10 h/sup -1/, AHI<15h/sup -1/, AHI<30h/sup -1/). Results showed that TH/sub adaptive/ outperformed TH/sub median/ on selecting regular snores. Moreover, the outcome achieved with non-regular snores intensity features suggests that these carry key information on SAHS severity.

JTD Keywords: Practical, Experimental/ acoustic signal processing, Bioacoustics, Biomedical measurement, Diseases, Feature extraction, Medical signal processing, Patient diagnosis, Pneumodynamics, Sleep/ nonregular snore features, SAHS markers, Sleep apnea hypopnea syndrome, Overnight multichannel polysomnography, Snore mechanism

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

JTD Keywords: Membrane Stress Failure, Frog Skeletal-Muscle, Extracellular-Matrix, Glass-Transition, Energy Landscape, Actin-Filaments, Living Cell, Single, Traction, Cytoskeleton

The structural integrity of the alveolar monolayer, which is compromised during lung inflammation, is determined by the balance between cell-cell and cell-matrix tethering forces and the centripetal forces owing to cell viscoelasticity and contraction. Dexamethasone is an anti-inflammatory glucocorticoid with protective effects in lung injury. To determine the effects of Dexamethasone on the stiffness and contractility of alveolar epithelial cells. Cell stiffness (G') and average traction exerted by the cell (T) were measured by magnetic twisting cytometry and by traction microscopy, respectively. A549 cells were treated 24 h with Dexamethasone (1 mu M) or vehicle (control). G' and T were measured before and 5 min after challenge with the inflammatory mediator Thrombin (0.5 U/ml). Changes induced by Dexamethasone in actin cytoskeleton polymerization were assessed by the fluorescent ratio between F-actin and G-actin obtained by staining cells with phalloidin and DNase I. Dexamethasone significantly increased G' and T by 56% (n = 11; p < 0.01) and by 80% (n = 17; p < 0.05), respectively. Dexamethasone also increased F/G-actin ratio from 2.68 +/- 0.07 to 2.96 +/- 0.09 (n = 10; p < 0.05). The relative increase in stiffness and contraction induced by Thrombin in control cells was significantly (p < 0.05) reduced by Dexamethasone treatment: from 190 to 98% in G' and from 318 to 105% in T. The cytoskeleton remodelling and the increase in cell stiffness and contraction induced by Dexamethasone could account for its protective effect in the alveolar epithelium when subjected to inflammatory challenge.

JTD Keywords: Cell mechanics, Cytoskeleton, Magnetic twisting cytometry, Traction microscopy, Respiratory diseases