Publications

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

ASM deficiency in Niemann-Pick disease type A results in aberrant cellular accumulation of sphingomyelin, neuroinflammation, neurodegeneration, and early death. There is no available treatment because enzyme replacement therapy cannot surmount the blood-brain barrier (BBB). Nanocarriers (NCs) targeted across the BBB via transcytosis might help; yet, whether ASM deficiency alters transcytosis remains poorly characterized. We investigated this using model NCs targeted to intracellular adhesion molecule-1 (ICAM-1), transferrin receptor (TfR), or plasmalemma vesicle-associated protein-1 (PV1) in ASM-normal vs. ASM-deficient BBB models. Disease differentially changed the expression of all three targets, with ICAM-1 becoming the highest. Apical binding and uptake of anti-TfR NCs and anti-PV1 NCs were unaffected by disease, while anti-ICAM-1 NCs had increased apical binding and decreased uptake rate, resulting in unchanged intracellular NCs. Additionally, anti-ICAM-1 NCs underwent basolateral reuptake after transcytosis, whose rate was decreased by disease, as for apical uptake. Consequently, disease increased the effective transcytosis rate for anti-ICAM-1 NCs. Increased transcytosis was also observed for anti-PV1 NCs, while anti-TfR NCs remained unaffected. A fraction of each formulation trafficked to endothelial lysosomes. This was decreased in disease for anti-ICAM-1 NCs and anti-PV1 NCs, agreeing with opposite transcytosis changes, while it increased for anti-TfR NCs. Overall, these variations in receptor expression and NC transport resulted in anti-ICAM-1 NCs displaying the highest absolute transcytosis in the disease condition. Furthermore, these results revealed that ASM deficiency can differently alter these processes depending on the particular target, for which this type of study is key to guide the design of therapeutic NCs.© 2023. Controlled Release Society.

JTD Keywords: asm deficiency, blood-brain barrier, delivery, determines, drug, endocytosis, enzymes, icam-1, lysosomal storage disease, mechanisms, nanoparticles, natural-history, niemann-pick disease type a, pv-1, receptor-mediated transcytosis, trafficking, transferrin receptor, Asm deficiency, Blood-brain barrier, Blood–brain barrier, Drug carriers, Drug nanocarriers, Humans, Icam-1, Icam-1-targeted nanocarriers, Intercellular adhesion molecule-1, Lysosomal storage disease, Niemann-pick disease type a, Niemann-pick disease, type a, Niemann-pick diseases, Pv-1, Receptor-mediated transcytosis, Transferrin receptor

Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disease diagnosed in childhood. It is a progressive and wasting disease, characterized by a degeneration of skeletal and cardiac muscles caused by the lack of dystrophin protein. The absence of this crucial structural protein leads to sarcolemmal fragility, resulting in muscle fiber damage during contraction. Despite ongoing efforts, there is no cure available for DMD patients. One of the primary challenges is the limited efficacy of current preclinical tools, which fail in modeling the biological complexity of the disease. Human-based three-dimensional (3D) cell culture methods appear as a novel approach to accelerate preclinical research by enhancing the reproduction of pathophysiological processes in skeletal muscle. In this work, we developed a patient-derived functional 3D skeletal muscle model of DMD that reproduces the sarcolemmal damage found in the native DMD muscle. These bioengineered skeletal muscle tissues exhibit contractile functionality, as they responded to electrical pulse stimulation. Sustained contractile regimes induced the loss of myotube integrity, mirroring the pathological myotube breakdown inherent in DMD due to sarcolemmal instability. Moreover, damaged DMD tissues showed disease functional phenotypes, such as tetanic fatigue. We also evaluated the therapeutic effect of utrophin upregulator drug candidates on the functionality of the skeletal muscle tissues, thus providing deeper insight into the real impact of these treatments. Overall, our findings underscore the potential of bioengineered 3D skeletal muscle technology to advance DMD research and facilitate the development of novel therapies for DMD and related neuromuscular disorders.

JTD Keywords: 3d cell culture, disease modeling, drug testing, duchenne muscular dystrophy, sarcolemmal damage, skeletal muscle, 3d cell culture, Animal-models, Disease modeling, Dmso, Drug testing, Duchenne muscular dystrophy, Gene, Humans, Image, Mechanisms, Muscle fibers, skeletal, Muscle, skeletal, Muscular dystrophy, duchenne, Myocardium, Sarcolemmal damage, Skeletal muscle, Tissue engineering, Utrophin

Prior studies demonstrated that encapsulation in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) enhanced the delivery of enzymes used for replacement therapy (ERT) of lysosomal storage disorders (LSDs). This study examined how the copolymer lactide:glycolide ratio impacts encapsulation, physicochemical characteristics, stability, and release under lysosomal conditions. Hyaluronidase, deficient in mucopolysaccharidosis IX, was encapsulated in NPs synthesized using 50:50, 60:40, or 75:25 lactide:glycolide copolymers. All NPs had diameters compatible with cellular transport (≤168 nm) and polydispersity indexes (≤0.16) and ζ-potentials (≤-35 mV) compatible with colloidal stability. Yet, their encapsulation efficiency varied, with 75:25 NPs and 60:40 NPs having the lowest and highest EE, respectively (15% vs. 28%). Under lysosomal conditions, the 50:50 copolymer degraded fastest (41% in 1 week), as expected, and the presence of a targeting antibody coat did not alter this result. Additionally, 60:40 NPs destabilized fastest (<1 week) because of their smaller diameter, and 75:25 NPs did not destabilize in 4 weeks. All formulations presented burst release under lysosomal conditions (56-78% of the original load within 30 min), with 50:50 and 60:40 NPs releasing an additional small fraction after week 1. This provided 4 weeks of sustained catalytic activity, sufficient to fully degrade a substrate. Altogether, the 60:40 NP formulation is preferred given its higher EE, and 50:50 NPs represent a valid alternative, while the highest stability of 75:25 NPs may impair lysosomes. These results can guide future studies aiming to translate PLGA NP-based ERT for this and other LSDs.

JTD Keywords: biodegradation, copolymer ratio, degradation, drug-delivery, emulsification, enzyme release, enzyme replacement therapy, hyaluronidase, mechanisms, microspheres, nanoparticle stability, poly(lactide-co-glycolide) nanoparticles, size, sphingomyelinase, transport, Central-nervous-system, Copolymer ratio, Enzyme release, Enzyme replacement therapy, Hyaluronidase, Lysosomal storage disorder, Nanoparticle stability, Poly(lactide-co-glycolide) nanoparticles

The Bin/amphiphysin/Rvs (BAR) superfamily proteins have a crescent binding domain and bend biomembranes along the domain axis. However, their anisotropic bending rigidities and spontaneous curvatures have not been experimentally determined. Here, we estimated these values from the bound protein densities on tethered vesicles using a mean-field theory of anisotropic bending energy and orientation-dependent excluded volume. The dependence curves of the protein density on the membrane curvature are fitted to the experimental data for the I-BAR and N-BAR domains reported by C. Prevost et al. Nat. Commun., 2015, 6, 8529 and F.-C. Tsai et al. Soft Matter, 2021, 17, 4254-4265, respectively. For the I-BAR domain, all three density curves of different chemical potentials exhibit excellent fits with a single parameter set of anisotropic bending energy. When the classical isotropic bending energy is used instead, one of the curves can be fitted well, but the others exhibit large deviations. In contrast, for the N-BAR domain, two curves are not well fitted simultaneously the anisotropic model, although it is significantly improved compared to the isotropic model. This deviation likely suggests a cluster formation of the N-BAR domains.

JTD Keywords: Membrane-mediated interactions,elastic properties,bar,shape,mechanisms,inclusions,generation,polymers,driven,bod

Sprouting angiogenesis is a core biological process critical to vascular development. Its accurate simulation, relevant to multiple facets of human health, is of broad, interdisciplinary appeal. This study presents an in-silico model replicating a microfluidic assay where endothelial cells sprout into a biomimetic extracellular matrix, specifically, a large-pore, low-concentration fibrin-based porous hydrogel, influenced by chemotactic factors. We introduce a novel approach by incorporating the extracellular matrix and chemotactic factor effects into a unified term using a single parameter, primarily focusing on modelling sprouting dynamics and morphology. This continuous model naturally describes chemotactic-induced sprouting with no need for additional rules. In addition, we extended our base model to account for matrix sensing and degradation, crucial aspects of angiogenesis. We validate our model via a hybrid in-silico experimental method, comparing the model predictions with experimental results derived from the microfluidic setup. Our results underscore the intricate relationship between the extracellular matrix structure and angiogenic sprouting, proposing a promising method for predicting the influence of the extracellular matrix on angiogenesis.Copyright © 2023 Ferre-Torres, Noguera-Monteagudo, Lopez-Canosa, Romero-Arias, Barrio, Castaño and Hernandez-Machado.

JTD Keywords: angiogenesis, biomimmetic, chemotaxis, endothelial cells, filopodia, growth, in silico model, mathematical models, mechanisms, metalloproteinase, migration, morphogenesis, phase field, pore-size, simulation, Angiogenesis, Biomimmetic, Chemotaxis, Endothelial cells, Extracellular matrix, In silico model, Mathematical models, Phase field, Tip cells

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.Copyright © 2023 Liang, Nilsson, Kragh, Hedal, Alcàcer-Almansa, Kiilerich, Andersen and Tolker-Nielsen.

JTD Keywords: aggregates, antibiotic tolerance, biofilm formation, extracellular matrix, genome, growth, lungs, molecular-mechanisms, mutations, polysaccharide, pseudomonas aeruginosa, psl, system, Aggregates, Antibiotic tolerance, Biofilm, Extracellular matrix, Pseudomonas aeruginosa, Small-colony variants

Transcriptomics and phosphoproteomics were carried out in the cerebral cortex of B6.Cg-Mapttm1(EGFP)Klt (tau knockout: tau-KO) and wild-type (WT) 12 month-old mice to learn about the effects of tau ablation. Compared with WT mice, tau-KO mice displayed reduced anxiety-like behavior and lower fear expression induced by aversive conditioning, whereas recognition memory remained unaltered. Cortical transcriptomic analysis revealed 69 downregulated and 105 upregulated genes in tau-KO mice, corresponding to synaptic structures, neuron cytoskeleton and transport, and extracellular matrix components. RT-qPCR validated increased mRNA levels of col6a4, gabrq, gad1, grm5, grip2, map2, rab8a, tubb3, wnt16, and an absence of map1a in tau-KO mice compared with WT mice. A few proteins were assessed with Western blotting to compare mRNA expression with corresponding protein levels. Map1a mRNA and protein levels decreased. However, β-tubulin III and GAD1 protein levels were reduced in tau-KO mice. Cortical phosphoproteomics revealed 121 hypophosphorylated and 98 hyperphosphorylated proteins in tau-KO mice. Deregulated phosphoproteins were categorized into cytoskeletal (n = 45) and membrane proteins, including proteins of the synapses and vesicles, myelin proteins, and proteins linked to membrane transport and ion channels (n = 84), proteins related to DNA and RNA metabolism (n = 36), proteins connected to the ubiquitin-proteasome system (UPS) (n = 7), proteins with kinase or phosphatase activity (n = 21), and 22 other proteins related to variegated pathways such as metabolic pathways, growth factors, or mitochondrial function or structure. The present observations reveal a complex altered brain transcriptome and phosphoproteome in tau-KO mice with only mild behavioral alterations.

JTD Keywords: computational platform, conformational-changes, cytoskeleton, disease, expression, isoforms, mechanisms, mice, phosphoproteomics, phosphorylation, synapse, tau-ko, tauopathies, transcriptomics, Tau-ko, Tau-protein, Transcriptomics

Development of liver fibrosis is paralleled by contraction of hepatic stellate cells (HSCs), the main profibrotic hepatic cells. Yet, little is known about the interplay of neprilysin (NEP) and its substrate neuropeptide Y (NPY), a potent enhancer of contraction, in liver fibrosis. We demonstrate that HSCs are the source of NEP. Importantly, NPY originates majorly from the splanchnic region and is cleaved by NEP in order to terminate contraction. Interestingly, NEP deficiency (Nep-/-) showed less fibrosis but portal hypertension upon liver injury in two different fibrosis models in mice. We demonstrate the incremental benefit of Nep-/- in addition to AT1R blocker (ARB) or ACE inhibitors for fibrosis and portal hypertension. Finally, oral administration of Entresto, a combination of ARB and NEP inhibitor, decreased hepatic fibrosis and portal pressure in mice. These results provide a mechanistic rationale for translation of NEP-AT1R-blockade in human liver fibrosis and portal hypertension.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: activation, cirrhosis, cirrhotic rats, cp: cell biology, expression, hepatic stellate cell, identification, inhibition, mechanisms, modulation, neprilysin, neuropeptide y, neuropeptide y receptor 1, portal hypertension, portal-hypertension, web server, Renin-angiotensin system

Escherichia coli is one of the most common members of the intestinal microbiota. Many of its strains are associated with various inflammatory infections, including urinary or gut infections, especially when displaying antibiotic resistance or in patients with suppressed immune systems. According to recent reports, the biofilm-forming potential of E. coli is a crucial factor for its increased resistance against antibiotics. To overcome the limitations of using antibiotics against resistant E. coli strains, the world is turning once more towards bacteriophage therapy, which is becoming a promising candidate amongst the current personalized approaches to target different bacterial infections. Although matured and persistent biofilms pose a serious challenge to phage therapy, they can still become an effective alternative to antibiotic treatment. Here, we assess the efficiency of clinically isolated phages in phage therapy against representative clinical uropathogenic and invasive biofilm-forming E. coli strains. Our results demonstrate that irrespective of host specificity, bacteriophages producing clear plaques with a high burst size, and exhibiting depolymerizing activity, are good candidates against biofilm-producing E. coli pathogens as verified from our in vitro and in vivo experiments using Galleria mellonella where survival was significantly increased for phage-therapy-treated larvae.

JTD Keywords: antibiotic resistance, assay, bacteriophage, bacteriophages, biofilm-forming potential, infection, inflammatory infections, mechanisms, Galleria-mellonella, Intestinal microflora

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young’s Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

JTD Keywords: atomic force microscopy, cells, deposition, extracellular matrix, idiopathic pulmonary fibrosis, mechanisms, mechanosensing, membranes, micromechanical properties, pathogenesis, stiffness, tissues, viscoelasticity, Extracellular matrix, Induced lung fibrosis, Mechanosensing

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.Copyright © 2023 Renau-Mínguez, Herrero-Abadía, Ruiz-Rodriguez, Sentandreu, Torrents, Chiner-Oms, Torres-Puente, Comas, Julián and Coscolla.

JTD Keywords: antimicrobial susceptibility, bcg, identification, immunogenic, non-pathogenic, nontuberculous mycobacteria, resistance mechanisms, strain, therapeutic, Diversity, Immunogenic, Non-pathogenic, Nontuberculous mycobacteria, Therapeutic

Glycans are ubiquitously expressed sugars, coating the cell and protein surfaces. They are found on many proteins as either short and branched chains or long chains sticking out from special membrane proteins, known as proteoglycans. This sugar cushion, the glycocalyx, modulates specific interactions and protects the cell. Here it is shown that both the expression of proteoglycans and the glycans expressed on the surface of both the host and virus proteins have a critical role in modulating viral attachment to the cell. A mathematical model using SARS-Cov-2 as an archetypical virus to study the glycan role during infection is proposed. It is shown that this occurs via a tug-of-war of forces. On one side, the multivalent molecular recognition that viral proteins have toward specific host glycans and receptors. On the other side, the glycan steric repulsion that a virus must overcome to approach such specific receptors. By balancing both interactions, viral tropism can be predicted. In other words, the authors can map out the cells susceptible to virus infection in terms of receptors and proteoglycans compositions.© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.

JTD Keywords: binding, entry, glycocalyx, mechanisms, multiplexing, multivalency, nanoparticles, recognition, super-selectivity, viral infectivity, Functional receptor, Glycans, Glycocalyx, Multiplexing, Multivalency, Nanoparticles, Super-selectivity, Viral infectivity

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.© 2022. Published by The Company of Biologists Ltd.

JTD Keywords: alpha-v-beta-3, arginine-glycine-aspartic acid, chondrogenesis, dynamics, expression, fibronectin, gastrulation, involvement, mechanisms, mesenchymal condensation, model, nanopatterned substrates, rgd, Arginine-glycine-aspartic acid, Cell migration, Chondrogenesis, Mesenchymal condensation, N-cadherin, Nanopatterned substrates, Rgd

Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

JTD Keywords: activation, angiogenesis, dynamics, flow, forces, image, mechanisms, vinculin, Angiogenesis, Cell polarity, Fluid shear, Mechanobiology, Morphogenesis, Shear stress

Abstract Nerves are a component of the tumor microenvironment contributing to cancer progression, but the role of cells from nerves in facilitating cancer invasion remains poorly understood. Here we show that Schwann cells (SCs) activated by cancer cells collectively function as Tumor Activated Schwann cell Tracks (TASTs) that promote cancer cell migration and invasion. Non-myelinating SCs form TASTs and have cell gene expression signatures that correlate with diminished survival in patients with pancreatic ductal adenocarcinoma. In TASTs, dynamic SCs form tracks that serve as cancer pathways and apply forces on cancer cells to enhance cancer motility. These SCs are activated by c-Jun, analogous to their reprogramming during nerve repair. This study reveals a mechanism of cancer cell invasion that co-opts a wound repair process and exploits the ability of SCs to collectively organize into tracks. These findings establish a novel paradigm of how cancer cells spread and reveal therapeutic opportunities.

JTD Keywords: dissemination, escape, mechanisms, progression, Perineural invasion

Chronic use of cannabis leads to both motor deficits and the downregulation of CB1 receptors (CB1R) in the cerebellum. In turn, cerebellar damage is often related to impairments in motor learning and control. Further, a recent motor learning task that measures cerebellar-dependent adaptation has been shown to distinguish well between healthy subjects and chronic cannabis users. Thus, the deteriorating effects of chronic cannabis use in motor performance point to cerebellar adaptation as a key process to explain such deficits. We review the literature relating chronic cannabis use, the endocannabinoid system in the cerebellum, and different forms of cerebellar-dependent motor learning, to suggest that CB1R downregulation leads to a generalized underestimation and misprocessing of the sensory errors driving synaptic updates in the cerebellar cortex. Further, we test our hypothesis with a computational model performing a motor adaptation task and reproduce the behavioral effect of decreased implicit adaptation that appears to be a sign of chronic cannabis use. Finally, we discuss the potential of our hypothesis to explain similar phenomena related to motor impairments following chronic alcohol dependency. © 2022

JTD Keywords: adaptation, addiction, alcohol-abuse, cerebellum, chronic cannabis use, cognition, deficits, endocannabinoid system, error processing, explicit, modulation, motor learning, release, synaptic plasticity, Adaptation, Adaptation, physiological, Alcoholism, Article, Behavioral science, Cannabinoid 1 receptor, Cannabis, Cannabis addiction, Cerebellum, Cerebellum cortex, Cerebellum disease, Chronic cannabis use, Computer model, Down regulation, Endocannabinoid, Endocannabinoid system, Endocannabinoids, Error processing, Hallucinogens, Human, Humans, Motor dysfunction, Motor learning, Nerve cell plasticity, Nonhuman, Physiology, Psychedelic agent, Purkinje-cells, Regulatory mechanism, Sensation, Sensory dysfunction, Sensory error processing impairment, Synaptic transmission, Task performance

Biomolecular gradients are widely present in multiple biological processes. Historically they were reproduced in vitro by using micropipettes, Boyden and Zigmond chambers, or hydrogels. Despite the great utility of these setups in the study of gradient-related problems such as chemotaxis, they face limitations when trying to translate more complex in vivo-like scenarios to in vitro systems. In the last 20 years, the advances in manufacturing of micromechanical systems (MEMS) had opened the possibility of applying this technology to biology (BioMEMS). In particular, microfluidics has proven extremely efficient in setting-up biomolecular gradients which are stable, controllable, reproducible and at length scales that are relevant to cells. In this chapter, we give an overview of different methods to generate molecular gradients using microfluidics, then we discuss the different steps of the pipeline to fabricate a gradient generator microfluidic device, and at the end, we show an application example of the fabrication of a microfluidic device that can be used to generate a surface-bound biomolecular gradient.© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.

JTD Keywords: biomems, gradient, microfluidics, model, nanotechnology, proteins, Biomems, Gradient, Mechanisms, Microfabrication, Microfluidics, Nanotechnology

Artificial control of neuronal activity enables the study of neural circuits and restoration of neural functions. Direct, rapid, and sustained photocontrol of intact neurons could overcome the limitations of established electrical stimulation such as poor selectivity. We have developed fast photoswitchable ligands of glutamate receptors (GluRs) to enable neuronal control in the auditory system. The new photoswitchable ligands induced photocurrents in untransfected neurons upon covalently tethering to endogenous GluRs and activating them reversibly with visible light pulses of a few milliseconds. As a proof of concept of these molecular prostheses, we applied them to the ultrafast synapses of auditory neurons of the cochlea that encode sound and provide auditory input to the brain. This drug-based method afforded the optical stimulation of auditory neurons of adult gerbils at hundreds of hertz without genetic manipulation that would be required for their optogenetic control. This indicates that the new photoswitchable ligands are also applicable to the spatiotemporal control of fast spiking interneurons in the brain.

JTD Keywords: Acid, Azobenzene, Glutamate-receptor, Ion channels, Mechanisms, Nerve, Optical switches, Release, Stimulation

Over the most recent decades, the development of new biological platforms to study disease progression and drug efficacy has been of great interest due to the high increase in the rate of neurodegenerative diseases (NDDs). Therefore, blood-brain barrier (BBB) as an organ-on-a-chip (OoC) platform to mimic brain-barrier performance could offer a deeper understanding of NDDs as well as a very valuable tool for drug permeability testing for new treatments. A very attractive improvement of BBB-oC technology is the integration of detection systems to provide continuous monitoring of biomarkers in real time and a fully automated analysis of drug permeably, rendering more efficient platforms for commercialization. In this Perspective, an overview of the main BBB-oC configurations is introduced and a critical vision of the BBB-oC platforms integrating electronic read out systems is detailed, indicating the strengths and weaknesses of current devices, proposing the great potential for biosensors integration in BBB-oC. In this direction, we name potential biomarkers to monitor the evolution of NDDs related to the BBB and/or drug cytotoxicity using biosensor technology in BBB-oC.

JTD Keywords: biosensors, blood−brain barrier (bbb), neurodegenerative diseases (ndds), organ-on-a-chip (ooc), Bbb, Biosensors, Blood-brain barrier (bbb), Electrical-resistance, Electrochemical biosensors, Endothelial-cells, In-vitro model, Matrix metalloproteinases, Mechanisms, Neurodegenerative diseases (ndds), Organ-on-a-chip (ooc), Permeability, Stress, Transendothelial electrical resistance (teer), Transepithelial, Transepithelial/transendothelial electrical resistance (teer), Transport

We studied how anisotropic proteins are orientationally ordered and change the radius of membrane tubes using mean-field theory with an orientation-dependent excluded volume interaction.

JTD Keywords: bar, density, driven, generation, inclusions, invagination, mechanisms, monte-carlo, tubulation, Mediated aggregation

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin

The use of broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) has been shown to be a promising therapeutic modality in the prevention of HIV infection. Understanding the b12-gp120 binding mechanism under physiological conditions may assist the development of more broadly effective antibodies. In this work, the main conformations and interactions between the receptor-binding domain (RBD) of spike glycoprotein gp120 of HIV-1 and the IgG1-b12 mAb are studied. Accelerated molecular dynamics (aMD) and ab initio hybrid molecular dynamics have been combined to determine the most persistent interactions between the most populated conformations of the antibody-antigen complex under physiological conditions. The results show the most persistent receptor-binding mapping in the conformations of the antibody-antigen interface in solution. The binding-free-energy decomposition reveals a small enhancement in the contribution played by the CDR-H3 region to the b12-gp120 interface compared to the crystal structure.

JTD Keywords: antibody, complex, functionals, gp120 envelope glycoprotein, hiv, immunodeficiency-virus, noncovalent interactions, simulations, software integration, Ab initio, Accelerated molecular dynamics, Accelerated molecular-dynamics, Antibodies, Antigens, Binding energy, Binding mechanisms, Computational studies, Crystal structure, Diseases, Free energy, Hiv infection, Human immunodeficiency virus, Molecular dynamics, Neutralizing antibodies, Physiological condition, Physiology, Receptor-binding domains, Therapeutic modality, Viruses

Atmospheric pressure plasma jets have been shown to impact several cancer cell lines, both in vitro and in vivo. These effects are based on the biochemistry of the reactive oxygen and nitrogen species generated by plasmas in physiological liquids, referred to as plasma-conditioned liquids. Plasma-conditioned media are efficient in the generation of reactive species, inducing selective cancer cell death. However, the concentration of reactive species generated by plasma in the cell culture media of different cell types can be highly variable, complicating the ability to draw precise conclusions due to the differential sensitivity of different cells to reactive species. Here, we compared the effects of direct and indirect plasma treatment on non-malignant bone cells (hOBs and hMSCs) and bone cancer cells (SaOs-2s and MG63s) by treating the cells directly or exposing them to previously treated cell culture medium. Biological effects were correlated with the concentrations of reactive species generated in the liquid. A linear increase in reactive species in the cell culture medium was observed with increased plasma treatment time independent of the volume treated. Values up to 700 µM for H2O2 and 140 µM of NO2− were attained in 2 mL after 15 min of plasma treatment in AdvDMEM cell culture media. Selectivity towards bone cancer cells was observed after both direct and indirect plasma treatments, leading to a decrease in bone cancer cell viability at 72 h to 30% for the longest plasma treatment times while maintaining the survival of non-malignant cells. Therefore, plasma-conditioned media may represent the basis for a potentially novel non-invasive technique for bone cancer therapy.

JTD Keywords: expression, in-vitro, jet, mechanisms, nitrate, nitrite, osteosarcoma cells, reactive oxygen, Cold atmospheric plasma

An amine functionalized C3-symmetric benzotrithiophene (BTT) monomer has been designed and synthetized in order to form pH responsive one-dimensional supramolecular polymers in aqueous media. While most of the reported studies looked at the effect of pH on the size of the aggregates, herein, a detailed mechanistic study is reported, carried out upon modifying the pH to trigger the formation of positively charged ammonium groups. A dramatic and reversible change in the polymerization mechanism and size of the supramolecular fibers is observed and ascribed to the combination of Coulombic repulsive forces and higher monomer solubility. Furthermore, the induced frustrated growth of the fibers is further employed to finely control the one-dimensional supramolecular polymerisation and copolymerization processes.

JTD Keywords: dynamics, ph responsivity, polymerization mechanism, self-assembly, supramolecular chemistry, supramolecular polymers, Ph responsivity, Polymerization mechanism, Polymers, Self-assembly, Supramolecular chemistry, Supramolecular polymers

Background: In recent decades, gold nanoparticle (Au NP)-based cancer therapy has been heavily debated. The physico-chemical properties of AuNPs can be exploited in photothermal therapy, making them a powerful tool for selectively killing cancer cells. However, the synthetic side products and capping agents often induce a strong activation of the inflammatory pathways of macrophages, thus limiting their further applications in vivo. Methods: Here, we described a green method to obtain stable polyphenol-capped AuNPs (Au NPs@polyphenols), as polyphenols are known for their anti-inflammatory and anticancer properties. These NPs were used in human macrophages to test key inflammation-related markers, such as NF-κB, TNF-α, and interleukins-6 and 8. The results were compared with similar NPs obtained by a traditional chemical route (without the polyphenol coating), proving the potential of Au NPs@polyphenols to strongly promote the shutdown of inflammation. This was useful in developing them for use as heat-synergized tools in the thermal treatment of two types of cancer cells, namely, breast cancer (MCF-7) and neuroblastoma (SH-SY5Y) cells. The cell viability, calcium release, oxidative stress, HSP-70 expression, mitochondrial, and DNA damage, as well as cytoskeleton alteration, were evaluated. Results: Our results clearly demonstrate that the combined strategy markedly exerts anticancer effects against the tested cancer cell, while neither of the single treatments (only heat or only NPs) induced significant changes. Conclusions: Au NP@polyphenols may be powerful agents in cancer treatment.

JTD Keywords: antioxidant, aunps, biocompatibility, biology, calcium, cancer, green synthesis, inflammation response, inhibition, interleukin-6, mechanisms, natural polyphenols, physico-chemical properties, polyphenols, size, thermal treatment, Aunps, Cancer, Green synthesis, Inflammation response, Nobilis l. leaves, Physico-chemical properties, Polyphenols, Thermal treatment

JTD Keywords: cardiac-hypertrophy, heart, mechanisms, Protein-kinase-d

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

JTD Keywords: Computational biology and bioinformatics, Genomics, Mechanisms of disease, Neurodegeneration, Systems biology

Prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing. Resulting fibrosis and portal hypertension, as a possible secondary event, may necessitate treatment. Overexpression of mouse renin in the transgenic rat model, TGR(mREN2)27, leads to spontaneous development of NAFLD. Therefore, we used TGR(mREN2)27 rats as a model of NAFLD where we hypothesized increased susceptibility and investigated fibrosis and portal hypertension and associated pathways. 12-week old TGR(mREN2)27 rats received either cholestatic (BDL) or toxic injury (CCl4 inhalation). Portal and systemic hemodynamic assessments were performed using microsphere technique with and without injection of the Janus-Kinase 2 (JAK2) inhibitor AG490 or the non-peptidic Ang(1-7) agonist, AVE0991. The extent of liver fibrosis was assessed in TGR(mREN2)27 and wild-type rats using standard techniques. Protein and mRNA levels of profibrotic, renin-angiotensin system components were assessed in liver and primary hepatic stellate cells (HSC) and hepatocytes. TGR(mREN2)27 rats developed spontaneous, but mild fibrosis and portal hypertension due to the activation of the JAK2/Arhgef1/ROCK pathway. AG490 decreased migration of HSC and portal pressure in isolated liver perfusions and in vivo. Fibrosis or portal hypertension after cholestatic (BDL) or toxic injury (CCl4) was not aggravated in TGR(mREN2)27 rats, probably due to decreased mouse renin expression in hepatocytes. Interestingly, portal hypertension was even blunted in TGR(mREN2)27 rats (with or without additional injury) by AVE0991. TGR(mREN2)27 rats are a suitable model of spontaneous liver fibrosis and portal hypertension but not with increased susceptibility to liver damage. After additional injury, the animals can be used to evaluate novel therapeutic strategies targeting Mas.

JTD Keywords: Mechanisms of disease, Molecular medicine

Rat model of Obstructive Sleep Apnea (OSA) is a realistic approach for studying physiological mechanisms involved in sleep. Rats are usually anesthetized and autonomic nervous system (ANS) could be blocked. This study aimed to assess the effect of anesthesia on ANS activity during OSA episodes. Seven male Sprague-Dawley rats were anesthetized intraperitoneally with urethane (1g/kg). The experiments were conducted applying airway obstructions, simulating 15s-apnea episodes for 15 minutes. Five signals were acquired: respiratory pressure and flow, SaO2, ECG and photoplethysmography (PPG). In total, 210 apnea episodes were studied. Normalized power spectrum of Pulse Rate Variability (PRV) was analyzed in the Low Frequency (LF) and High Frequency (HF) bands, for each episode in consecutive 15s intervals (before, during and after the apnea). All episodes showed changes in respiratory flow and SaO₂ signal. Conversely, decreases in the amplitude fluctuations of PPG (DAP) were not observed. Normalized LF presented extremely low values during breathing (median=7,67%), suggesting inhibition of sympathetic system due to anesthetic effect. Subtle increases of LF were observed during apnea. HRV and PPG analysis during apnea could be an indirect tool to assess the effect and deep of anesthesia.

JTD Keywords: electrocardiography, fluctuations, medical disorders, medical signal detection, medical signal processing, neurophysiology, photoplethysmography, pneumodynamics, sleep, ECG, SaO₂ flow, SaO₂ signal, airway obstructions, amplitude fluctuations, anesthesia effects, anesthetized nervous system, autonomic evaluation, autonomic nervous system, breathing, heart rate variability, high-frequency bands, low-frequency bands, male Sprague-Dawley rats, normalized power spectrum, obstructive sleep apnea, photoplethysmography, physiological mechanisms, pulse rate variability, rat model, respiratory flow, respiratory pressure, signal acquisition, sympathetic system inhibition, time 15 min, time 15 s, Abstracts, Atmospheric modeling, Computational modeling, Electrocardiography, Rats, Resonant frequency

In this work, we propose an easy method to produce highly tunable gradients of covalently bound proteins on topographically modified poly(methyl methacrylate). We used a rnicrofluidic approach to obtain linear gradients with high slope (0.5 pmol.cm(-2).mm(-1)), relevant at the single-cell level. These protein gradients were characterized using fluorescence microscopy and surface plasmon resonance. Both experimental results and theoretical modeling on the protein gradients generated have proved them to be highly reproducible, stable up to 7 days, and easily tunable. This method enables formation of versatile cell culture platforms combining both complex biochemical and physical cues in an attempt to approach in vitro cell culture methods to in vivo cellular microenvironments.

JTD Keywords: Cell-migration, Microfluidic channel, Surface, Streptavidin, Molecules, Topography, Mechanisms, Generation, Responses, Guidance

High altitude periodic breathing (PB) shares some common pathophysiologic aspects with sleep apnea, Cheyne-Stokes respiration and PB in heart failure patients. Methods that allow quantifying instabilities of respiratory control provide valuable insights in physiologic mechanisms and help to identify therapeutic targets. Under the hypothesis that high altitude PB appears even during physical activity and can be identified in comparison to visual analysis in conditions of low SNR, this study aims to identify PB by characterizing the respiratory pattern through the respiratory volume signal. A number of spectral parameters are extracted from the power spectral density (PSD) of the volume signal, derived from respiratory inductive plethysmography and evaluated through a linear discriminant analysis. A dataset of 34 healthy mountaineers ascending to Mt. Muztagh Ata, China (7,546 m) visually labeled as PB and non periodic breathing (nPB) is analyzed. All climbing periods within all the ascents are considered (total climbing periods: 371 nPB and 40 PB). The best crossvalidated result classifying PB and nPB is obtained with Pm (power of the modulation frequency band) and R (ratio between modulation and respiration power) with an accuracy of 80.3% and area under the receiver operating characteristic curve of 84.5%. Comparing the subjects from 1_st and 2_nd ascents (at the same altitudes but the latter more acclimatized) the effect of acclimatization is evaluated. SaO₂ and periodic breathing cycles significantly increased with acclimatization (p-value <; 0.05). Higher Pm and higher respiratory frequencies are observed at lower SaO₂, through a significant negative correlation (p-value <; 0.01). Higher Pm is observed at climbing periods visually labeled as PB with >; 5 periodic breathing cycles through a significant positive correlation (p-value <; 0.01). Our data demonstrate that quantification of the respiratory volum- signal using spectral analysis is suitable to identify effects of hypobaric hypoxia on control of breathing.

JTD Keywords: Frequency domain analysis, Frequency modulation, Heart, Sleep apnea, Ventilation, Visualization, Cardiology, Medical disorders, Medical signal processing, Plethysmography, Pneumodynamics, Sensitivity analysis, Sleep, Spectral analysis, Cheyne-Stokes respiration, Climbing periods, Dataset, Heart failure patients, High altitude PB, High altitude periodic breathing, Hypobaric hypoxia, Linear discriminant analysis, Pathophysiologic aspects, Physical activity, Physiologic mechanisms, Power spectral density, Receiver operating characteristic curve, Respiratory control, Respiratory frequency, Respiratory inductive plethysmography, Respiratory pattern, Respiratory volume signal, Sleep apnea, Spectral analysis, Spectral parameters

This work presents the estimation of a likelihood map for the location of a source of chemical plume using multiple mobile sensors and Bayesian Inference. Previously described methods use a single sensor and just binary detections (concentrations above or below a certain threshold). The main contribution of this work is to extend previous proposals to use concentration information while at the same time being robust against the presence of background signals. The algorithm has two parts. The first part, concerning Adaptive Background Estimation, uses robust statistics measurements to estimate the background level despite the intermittent presence of high concentrations due to plume statistics. The second part of the algorithm estimates likelihood functions for background and for condition plus plume. Then, the algorithm sequentially builds a likelihood probability map for the location of the source. The algorithm allows the use of multiple mobile sensors. The simulation results demonstrate that our algorithm estimates better the source location and is much more robust in the presence of false alarms.

JTD Keywords: Sensors, Inference mechanisms, Probability, Simulation

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +/- 0.58 (m +/- SE) and 1.00 +/- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +/- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +/- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

JTD Keywords: Induced acute lung, Sleep-apnea, Intermitent hypoxia, Cardiovascular-disease, Progenito Cells, Rat model, Inflammation, Mechanisms, Repair, Blood