Publications

Electrochemical sensors are sophisticated devices capable of detecting a wide range of chemical compounds with exceptional sensitivity and efficiency. Their importance is particularly pronounced in biomedical applications, where the rapid and accurate detection of biomolecules such as dopamine (DA), glucose (G), and nicotinamide adenine dinucleotide (NADH) is crucial for early diagnosis and disease management. These biomarkers are key in monitoring and managing conditions like diabetes, Parkinson and Alzheimer diseases, and bacterial infections. This review provides a comprehensive overview of electrochemical biosensors, detailing the methodologies commonly used by researchers and the latest technological advancements that enable more efficient device development. In this regard, the focus is on the impact and trends of various materials utilized in the fabrication of electrochemical biosensors, including conducting polymers, ceramics, and carbon-based materials. By examining the state of the art, we explore how these materials contribute to enhanced performance and reliability. Furthermore, while the development of highly selective and sensitive nanocomposites has been a primary focus in the field, this review also highlights efforts toward creating cost-effective biosensors with rapid prototyping capabilities. Such innovations aim to maintain high efficacy in electrochemical detection while making advanced diagnostics more accessible. In conclusion, this study aims to inform researchers and professionals about the evolving materials landscape in electrochemical biosensing, offering insights into the future directions of this critical technology.

JTD Keywords: Biomolecules, Biosensor, Dopamine, Electroactive materials, Electrochemical sensors, Electrode, Glucose biosensor, Nanoparticles, Nanosheets, Oxidas, Sensor, Temperature, Transition-metal carbides, Uric-acid

Chimeric antigen receptor (CAR)-T cells have made significant advancements in the field of adoptive immune cell therapies and the treatment of hematological malignancies. However, there are several drawbacks associated with the production and administration of these therapies. As a result, there has been interest in using natural killer (NK) cells to develop allogeneic CAR-NK cell therapies instead. While viral transduction is powerful for engineering T cells, NK cells have shown limited efficacy and high toxicity with this method. Therefore, efforts are being made to optimize non-viral transfection technologies for engineering NK cells. One such emerging technology is photoporation, which has demonstrated high efficiency and versatility for transfecting different immune cells. In this study, we evaluated the potential of nanoparticle-sensitized photoporation for genetic engineering of NK cells. Our findings show that both FD500 and eGFP mRNA can be successfully delivered into NK-92MI cells with high efficiency and low toxicity. When compared to state-of-the-art electroporation, photoporation proved to be more efficient, gentle, and capable of preserving the phenotype of NK-92MI cells. Overall, our work highlights the promising prospects of photoporation for NK cell engineering.

JTD Keywords: Cancer immunotherapie, Car, Cell engineering, Deliver, Messenger-rna, Nanoparticles, Natural killer cells, Natural-killer-cells, Photoporation, Polydopamine nanoparticles, T-cells

The development of smart biomedical devices as efficient tools in early diagnosis and therapy monitoring has recently witnessed unprecedented growth, becoming an emerging field in biomedical engineering. Sponges for endoluminal vacuum therapy, which are intended for transmitting negative pressure as trigger for tissue regeneration and for draining infections in anastomotic leakages, are massively used implants with very complex geometry and high risk of infection. In this work, commercial polyurethane (PU) sponges have been converted into smart biomedical devices by incorporating an electrochemical sensor to monitor the growth of bacteria. Such innovative approach, which allows to track the tissue healing process avoiding further infection development, has been performed applying a three-step process: 1) activation of PU using low pressure oxygen plasma; 2) incorporation of conducting polymer (CP) nanoparticles (NPs) at the surface of the activated PU by chemical oxidative polymerization; and 3) formation of a homogeneous electroactive coating using the CP NPs obtained in 2), as growth nuclei in an electrochemical polymerization. The functionalized PU sponge is able to monitor the bacteria growth in the surrounding media by detecting the concentration of nicotinamide adenine dinucleotide (NADH) from respiration reactions in the cytosol (i.e. bacteria do not have mitochondria). Conversely, respiration in normal eukaryotic cells takes place in the mitochondria, whose double membrane is not permeable to NADH. The sensing performance of the CP-coated PU sponges (limit of detection: 0.06 mM; sensitivity: 1.21 mA/cm2) has been determined in the lab using NADH solutions, while a proof of concept have been conducted using Escherichia coli bacteria cultures.

JTD Keywords: Conducting polymer, Desig, Electrochemical coating, Esophageal cancer, Nadh, Pedot, Polyurethane functionalization, Selective detection, Sponge functionalizatio

Clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this, diagnosis at asymptomatic stages of Alzheimer ' s disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment. Many studies have determined that the expression pattern of some miRNAs is dysregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrP C ) during disease progression. That is why, by means of cross studies of miRNAs up -regulated in AD with in silico identification of potential miRNAs-binding to 3 ' UTR of human PRNP gene, we selected miR-519a-3p for our study. Then, in vitro experiments were carried out in two ways. First, we validated miR-519a-3p target on 3 ' UTR- PRNP , and second, we analyzed the levels of PrP C expression after using of mimic technology on cell culture. In addition, RT-qPCR was performed to analyzed miR519a-3p expression in human cerebral samples of AD at different stages of disease evolution. Additionally, samples of other neurodegenerative diseases such as other non -AD tauopathies and several synucleinopathies were included in the study. Our results showed that miR-519a-3p overlaps with PRNP 3 ' UTR in vitro and promotes downregulation of PrP C . Moreover, miR-519a-3p was found to be up -regulated exclusively in AD samples from stage I to VI, suggesting its potential use as a novel label of preclinical stages of the disease.

JTD Keywords: Activation, Alzheimer's disease, Biomarke, Brain, Cellular prion protein, Developmental expression, Gen, Micrornas, Plasma, Prpc, Tau-aggregation

Micro/nanomotors (MNMs) have evolved from single self-propelled entities to versatile systems capable of performing one or multiple biomedical tasks. When single MNMs self-assemble into coordinated swarms, either under external control or triggered by chemical reactions, they offer advantages that individual MNMs cannot achieve. These benefits include intelligent multitasking and adaptability to changes in the surrounding environment. Here, we provide our perspective on the evolution of MNMs, beginning with the development of enzymatic MNMs since the first theoretical model was proposed in 2005. These enzymatic MNMs hold immense promise in biomedicine due to their advantages in biocompatibility and fuel availability. Subsequently, we introduce the design and application of single motors in biomedicine, followed by the control of MNM swarms and their biomedical applications. In the end, we propose viable solutions for advancing the development of MNM swarms and anticipate valuable insights into the creation of more intelligent and controllable MNM swarms for biomedical applications.; Micro/nanomotor swarms propelled by diverse mechanisms.

JTD Keywords: Active particles, Actuation, Behaviors, Biocompatibility, Biomedical applications, Coordination reactions, Design and application, Diffusion, External control, Medical applications, Micromotors, Motion, Nanomotors, Powered nanomotors, Propulsion, Self-assemble, Surrounding environment, Theoretical modeling, Versatile system, Viable solutions

Simple Summary Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies.Abstract Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

JTD Keywords: Alternative vasculature, Angiogenesis, Cells, Differentiation, Gb3, Neuroblastoma, Origin, Tumor-derived endothelial cells

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L-0 phases in a liquid-disordered L-d phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

JTD Keywords: Article, Artificial cells, Atomic force microscopy, Bicomponents, Bilayer, Bilayer membrane, Biochemical functionality, Biochemical process, Biological-membranes, Cell component, Cell membrane, Cellular parameters, Chemical interaction, Chemical structure, Chemistry, Cytology, Defined janus glycodendrimers, Dehydration, Dendrimer, Dendrimers, Dilution, Dimer, External agents, Fourier transform, Giant vesicles, Glycan, Glycans, Glycocalyx, Glycodendrimers, Janus dendrimer, Janus glycodendrimer, Lactose, Lateral organization, Lectin, Lipid rafts, Living organisms, Membrane damage, Membrane microdomain, Membrane microdomains, Membrane structure, Metabolism, Modulated phases, Molecule, Monomer, Nanoarrays, Oligosaccharide, Organization, Periodicity, Phase separation, Phase-separation, Phospholipids, Polysaccharide, Polysaccharides, Raft like domain, Relative humidity, Spatial segregation, Structure analysis, Sugars, Synthetic systems, Tetramer, Unclassified drug, Unilamellar vesicles, Water

Human pluripotent stem cell -derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single -cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA -approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

JTD Keywords: Adenylate kinase, Adult, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Autophagosome, Autophagy, Autophagy (cellular), Autosomal-dominant, Calcium homeostasis, Cilia, Cilium, Cohort analysis, Controlled study, Cyclic amp, Disease, Dominant polycystic kidney, Enzyme linked immunosorbent assay, Epithelium, Exon, Expression, Female, Food and drug administration, Framework, Generation, Growth, Hepatitis a virus cellular receptor 1, Human, Human cell, Humans, Immunohistochemistry, In vitro study, In vivo study, Kidney, Kidney organoid, Kidney polycystic disease, Male, Minoxidil, Mouse, Mutations, Nonhuman, Organoid, Organoids, Platelet derived growth factor beta receptor, Pluripotent stem-cells, Polycystic kidney diseases, Protein kinase lkb1, Renin, Sequestosome 1, Single cell analysis, Single cell rna seq, Small nuclear rna, Tunel assay, Upregulation, Western blotting, Whole exome sequencing

The last decade has witnessed great progress in the field of adoptive cell therapies, with the authorization of Kymriah (tisagenlecleucel) in 2017 by the Food and Drug Administration (FDA) as a crucial stepstone. Since then, five more CAR-T therapies have been approved for the treatment of hematological malignancies. While this is a great step forward to treating several types of blood cancers, CAR-T cell therapies are still associated with severe side-effects such as Graft-versus-Host Disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity. Because of this, there has been continued interest in Natural Killer cells which avoid these side-effects while offering the possibility to generate allogeneic cell therapies. Similar to T-cells, NK cells can be genetically modified to improve their therapeutic efficacy in a variety of ways. In contrast to T cells, viral transduction of NK cells remains inefficient and induces cytotoxic effects. Viral vectors also require a lengthy and expensive product development process and are accompanied by certain risks such as insertional mutagenesis. Therefore, non-viral transfection technologies are avidly being developed aimed at addressing these shortcomings of viral vectors. In this review we will present an overview of the potential of NK cells in cancer immunotherapies and the non-viral transfection technologies that have been explored to engineer them.Copyright © 2023 Elsevier Inc. All rights reserved.

JTD Keywords: adoptive cell therapy, cancer immunotherapy, immunotherapy, messenger-rna delivery, nanoparticle, nk cells, non -viral engineering, sonoporation, t-cell, transfection, ultrasound, Adoptive cell therapy, Cancer immunotherapy, Cell engineering, Natural-killer-cells, Nk cells, Non-viral engineering

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

JTD Keywords: extracellular vesicles, microfluidic platforms, micrornas, obesity, plasma, rnas, Biomarkers, Mirnas

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

JTD Keywords: drug delivery, icam-1, inflammation, lung injury, messenger-rna, migration, model, nanoparticles, neutrophils, pharmacokinetics, t-cells, white bloodcells, Adhesion molecules, Brain, Drug delivery, Inflammation, Nanoparticles, Pharmacokinetics, White blood cells

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

JTD Keywords: 3 & prime, alternative splicing, gene expression, meiosis, oocyte competence, program, rna, splicing, untranslated region processing, untranslated regions, 3′ untranslated region processing, 3′ untranslated regions, Alternative splicing, Expression, Gene expression, Human oocytes, Meiosis, Oocyte competence, Splicing

Background In mechanical thrombectomy (MT), distal access catheters (DACs) are tracked through the vascular anatomy to reach the occlusion site. The inability of DACs to reach the occlusion site has been reported as a predictor of unsuccessful recanalization. This study aims to provide insight into how to navigate devices through the vascular anatomy with minimal track forces, since higher forces may imply more risk of vascular injuries. Methods We designed an experimental setup to monitor DAC track forces when navigating through an in vitro anatomical model. Experiments were recorded to study mechanical behaviors such as tension buildup against vessel walls, DAC buckling, and abrupt advancements. A multiple regression analysis was performed to predict track forces from the catheters' design specifications. Results DACs were successfully delivered to the target M1 in 60 of 63 in vitro experiments (95.2%). Compared to navigation with unsupported DAC, the concomitant coaxial use of a microcatheter/microguidewire and microcatheter/stent retriever anchoring significantly reduced the track forces by about 63% and 77%, respectively (p<0.01). The presence of the braid pattern in the reinforcement significantly reduced the track forces regardless of the technique used (p<0.05). Combined coil and braid reinforcement configuration, as compared with coil alone, and a thinner distal wall were predictors of lower track force when navigating with unsupported DAC. Conclusions The use of microcatheter and stent retriever facilitate smooth navigation of DACs through the vascular tortuosity to reach the occlusion site, which in turn improves the reliability of tracking when positioning the DAC closer to the thrombus interface.

JTD Keywords: catheter, navigation, stroke, thrombectomy, Catheter, Navigation, Stroke, Thrombectomy, Vessel wall

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

JTD Keywords: antisense oligonucleotides, aon, blockmir, brain, expression, genes, mbnl, mir-218, mir-23b, mirna, muscleblind, myotonic dystrophy 1, phenotypes, proteins, type-1, Antisense oligonucleotides, Aon, Blockmir, Mbnl, Messenger-rna, Mir-218, Mir-23b, Mirna, Muscleblind, Myotonic dystrophy 1

Drug and gene delivery systems based on polymeric nanoparticles offer a greater efficacy and a reduced toxicity compared to traditional formulations. Recent studies have evidenced that their internalization, biodistribution and efficacy can be affected, among other factors, by their mechanical properties. Here, we analyze by means of Atomic Force Microscopy force spectroscopy how composition, surface functionalization and loading affect the mechanics of nanoparticles. For this purpose, nanoparticles made of Poly(lactic-co-glycolic) (PLGA) and Ethyl cellulose (EC) with different functionalizations and loading were prepared by nano-emulsion templating using the Phase Inversion Composition method (PIC) to form the nano-emulsions. A multiparametric nanomechanical study involving the determination of the Young's modulus, maximum deformation and breakthrough force was carried out. The obtained results showed that composition, surface functionalization and loading affect the nanomechanical properties in a different way, thus requiring, in general, to consider the overall mechanical properties after the addition of a functionalization or loading. A graphical representation method has been proposed enabling to easily identify mechanically equivalent formulations, which is expected to be useful in the development of soft polymeric nanoparticles for pre-clinical and clinical use.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: afm, atomic-force microscopy, cell, delivery-systems, drug-delivery, emulsification approach, internalization, mechanics of nanoparticles, nanomedicine, nanoparticle functionalization, particles, protein corona, size, young?s modulus, Afm, Loaded plga nanoparticles, Mechanics of nanoparticles, Nanomedicine, Nanoparticle functionalization, Polymeric nanoparticles, Young’s modulus

Prion-like domains (PrLDs) are intrinsically disordered regions (IDRs) of low sequence complexity with a similar composition to yeast prion domains. PrLDs-containing proteins have been involved in different organisms' regulatory processes. Regions of moderate amyloid propensity within IDRs have been shown to assemble autonomously into amyloid fibrils. These sequences tend to be rich in polar amino acids and often escape from the detection of classical bioinformatics screenings that look for highly aggregation-prone hydrophobic sequence stretches. We defined them as cryptic amyloidogenic regions (CARs) and recently developed an integrated database that collects thousands of predicted CARs in IDRs. CARs seem to be evolutionary conserved among disordered regions because of their potential to stablish functional contacts with other biomolecules. Here we have focused on identifying and characterizing CARs in prion-like proteins (pCARs) from plants, a lineage that has been poorly studied in comparison with other prionomes. We confirmed the intrinsic amyloid potential for a selected pCAR from Arabidopsis thaliana and explored functional enrichments and compositional bias of pCARs in plant prion-like proteins.Copyright © 2023 Pintado-Grima, Santos, Iglesias, Manglano-Artuñedo, Pallarès and Ventura.

JTD Keywords: aggregation, aromatic residues, bioinformatics, domains, functional interactions, identify proteins, plants, prediction, prion-like domains, q/n-rich, regulator, sup35, yeast, Bioinformatics, Cryptic amyloidogenic regions, Functional interactions, Plants, Prion-like domains, Rna-binding proteins

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: colonization, defines, human colon, mutations, plasticity, retrieval, stem-cells, subtypes, underlie, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Article, Cancer, Cancer growth, Cancer immunotherapy, Cancer inhibition, Cancer recurrence, Cancer staging, Cell, Cell adhesion, Cell migration, Cell population, Cell surface receptor, Cohort analysis, Colorectal cancer, Colorectal neoplasms, Colorectal tumor, Comprehensive molecular characterization, Controlled study, Crispr-cas9 system, Cytoskeleton, Disease exacerbation, Disease progression, Dynamics, Emp1 gene, Epithelial membrane protein-1, Extracellular matrix, Flow cytometry, Fluorescence intensity, Gene expression, Genetics, Human, Human cell, Humans, Immune response, Immunofluorescence, In situ hybridization, Marker gene, Metastasis potential, Mice, Minimal residual disease, Mouse, Neoplasm proteins, Neoplasm recurrence, local, Neoplasm, residual, Nonhuman, Pathology, Phenotype, Prevention and control, Protein, Receptors, cell surface, Single cell rna seq, Tumor, Tumor protein, Tumor recurrence

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form.Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.

JTD Keywords: adsorption, cryo-soft x-ray tomography, cytotoxicity, expression, flow cytometry, internalisation, intracellular calcium, magnesium, nano, nanomaterials, nanoparticles, proliferation, protein corona, ultrastructure, Calcium-phosphate nanoparticles, Cryo-soft x-ray tomography, Flow cytometry, Hydroxyapatite, Internalisation, Intracellular calcium, Nanoparticles

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active b1-integrin content on the surface of Cav1KO fibroblasts. FRAP analysis and endocytosis/recycling assays revealed that active b1-integrin is mostly endocytosed through the CLIC/GEEC pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven b1-integrin activation is lower in Cav1KO MEFs than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.© 2022, Lolo et al.

JTD Keywords: adhesion, alpha-v-beta-3, cell, integrin activation, internalization, kinase, mechanosensing, mediated endocytosis, mouse, stiffness, talin, trafficking, Animals, Caveolae, Cell adhesion, Cell biology, Fibroblasts, Integrin activation, Integrin beta1, Integrin recycling, Integrins, Mechanosensing, Membrane tension, Mice, Mouse, Stress, mechanical

Erythrina velutina is a Brazilian native tree of the Caatinga (a unique semiarid biome). It is widely used in traditional medicine showing anti-inflammatory and central nervous system modulating activities. The species is a rich source of specialized metabolites, mostly alkaloids and flavonoids. To date, genomic information, biosynthesis, and regulation of flavonoids remain unknown in this woody plant. As part of a larger ongoing research goal to better understand specialized metabolism in plants inhabiting the harsh conditions of the Caatinga, the present study focused on this important class of bioactive phenolics. Leaves and seeds of plants growing in their natural habitat had their metabolic and proteomic profiles analyzed and integrated with transcriptome data. As a result, 96 metabolites (including 43 flavonoids) were annotated. Transcripts of the flavonoid pathway totaled 27, of which EvCHI, EvCHR, EvCHS, EvCYP75A and EvCYP75B1 were identified as putative main targets for modulating the accumulation of these metabolites. The highest correspondence of mRNA vs. protein was observed in the differentially expressed transcripts. In addition, 394 candidate transcripts encoding for transcription factors distributed among the bHLH, ERF, and MYB families were annotated. Based on interaction network analyses, several putative genes of the flavonoid pathway and transcription factors were related, particularly TFs of the MYB family. Expression patterns of transcripts involved in flavonoid biosynthesis and those involved in responses to biotic and abiotic stresses were discussed in detail. Overall, these findings provide a base for the understanding of molecular and metabolic responses in this medicinally important species. Moreover, the identification of key regulatory targets for future studies aiming at bioactive metabolite production will be facilitated.

JTD Keywords: caatinga, erythrina velutina, flavonoids, molecular network, Arabidopsis, Caatinga, Classification, Discovery, Erythrina velutina, Flavonoids, Identification, Mass-spectrometry, Messenger-rna, Metabolism, Molecular network, Natural-products, Protein abundance, Transcriptome

Moderate exercise has well-founded benefits in cardiovascular health. However, increasing, yet controversial, evidence suggests that extremely trained athletes may not be protected from cardiovascular events as much as moderately trained individuals. In our rodent model, intensive but not moderate training promoted aorta and carotid stiffening and elastic lamina ruptures, tunica media thickening of intramyocardial arteries, and an imbalance between vasoconstrictor and relaxation agents. An up-regulation of angiotensin-converter enzyme, miR-212, miR-132, and miR-146b might account for this deleterious remodeling. Most changes remained after a 4-week detraining. In conclusion, our results suggest that intensive training blunts the benefits of moderate exercise. (C) 2022 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

JTD Keywords: atherosclerosis, cacs, coronary artery calcium score, cad, coronary artery disease, coronary artery disease, cv, cardiovascular, endurance exercise, extreme sport, mmp9, matrix metalloproteinase 9, no, nitric oxide, phe, phenylephrine, vsmc, vascular smooth muscle cell, Age, Atherosclerosis, Cacs, coronary artery calcium score, Cad, coronary artery disease, Coronary artery disease, Coronary atherosclerosis, Cv, cardiovascular, Disease, Endurance exercise, Extreme sport, Metalloproteinases, Micrornas, Mmp9, matrix metalloproteinase 9, No, nitric oxide, Phe, phenylephrine, Physical-activity, Prevalence, Rats, Relevance, Risk, Vascular stiffening, Vsmc, vascular smooth muscle cell

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

Nanomedicine involves the use of nanotechnology for clinical applications and holds promise to improve treatments. Recent developments offer new hope for cancer detection, prevention and treatment; however, being a heterogenous disorder, cancer calls for a more targeted treatment approach. Personalized Medicine (PM) aims to revolutionize cancer therapy by matching the most effective treatment to individual patients. Nanotheranostics comprise a combination of therapy and diagnostic imaging incorporated in a nanosystem and are developed to fulfill the promise of PM by helping in the selection of treatments, the objective monitoring of response and the planning of follow-up therapy. Although well-established imaging techniques, such as Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT), are primarily used in the development of theranostics, Optical Imaging (OI) offers some advantages, such as high sensitivity, spatial and temporal resolution and less invasiveness. Additionally, it allows for multiplexing, using multi-color imaging and DNA barcoding, which further aids in the development of personalized treatments. Recent advances have also given rise to techniques permitting better penetration, opening new doors for OI-guided nanotheranostics. In this review, we describe in detail these recent advances that may be used to design and develop efficient and specific nanotheranostics for personalized cancer drug delivery. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: 5-aminolevulinic acid, cancer, contrast agents, in-vivo, malignant gliomas, multifunctional nanoparticles, nanomedicine, optical imaging, ovarian-cancer, personalized medicine, quantum dots, silica nanoparticles, targeted probes, theranostics, Cancer, Nanomedicine, Optical imaging, Personalized medicine, Superparamagnetic iron-oxide, Theranostics

Background Cellular prion protein (PrP(C)) is a cell surface GPI-anchored protein, usually known for its role in the pathogenesis of human and animal prionopathies. However, increasing knowledge about the participation of PrP(C) in prion pathogenesis contrasts with puzzling data regarding its natural physiological role. PrP(C) is expressed in a number of tissues, including at high levels in the nervous system, especially in neurons and glial cells, and while previous studies have established a neuroprotective role, conflicting evidence for a synaptic function has revealed both reduced and enhanced long-term potentiation, and variable observations on memory, learning, and behavior. Such evidence has been confounded by the absence of an appropriate knock-out mouse model to dissect the biological relevance of PrP(C), with some functions recently shown to be misattributed to PrP(C) due to the presence of genetic artifacts in mouse models. Here we elucidate the role of PrP(C) in the hippocampal circuitry and its related functions, such as learning and memory, using a recently available strictly co-isogenic Prnp(0/0) mouse model (Prnp(ZH3/ZH3)). Results We performed behavioral and operant conditioning tests to evaluate memory and learning capabilities, with results showing decreased motility, impaired operant conditioning learning, and anxiety-related behavior in Prnp(ZH3/ZH3) animals. We also carried in vivo electrophysiological recordings on CA3-CA1 synapses in living behaving mice and monitored spontaneous neuronal firing and network formation in primary neuronal cultures of Prnp(ZH3/ZH3) vs wildtype mice. PrP(C) absence enhanced susceptibility to high-intensity stimulations and kainate-induced seizures. However, long-term potentiation (LTP) was not enhanced in the Prnp(ZH3/ZH3) hippocampus. In addition, we observed a delay in neuronal maturation and network formation in Prnp(ZH3/ZH3) cultures. Conclusion Our results demonstrate that PrP(C) promotes neuronal network formation and connectivity. PrP(C) mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion may underlie an epileptogenic-susceptible brain that fails to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.

JTD Keywords: anxiety, behavior, cellular prion protein, epilepsy, hippocampus, Anxiety, Behavior, Cellular prion protein, Developmental expression, Epilepsy, Gene-expression, Hippocampus, Kainate-induced seizures, Lacking, Ltp, Memory, Messenger-rna, Motor behavior, Mouse, Prp

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

Several studies have demonstrated the different characteristics of tau seeding and spreading following intracerebral inoculation in murine models of tau-enriched fractions of brain homogenates from AD and other tauopathies. The present study is centered on the importance of host tau in tau seeding and the molecular changes associated with the transformation of host tau into abnormal tau. The brains of three adult murine genotypes expressing different forms of tau—WT (murine 4Rtau), hTau (homozygous transgenic mice knock-out for murine tau protein and heterozygous expressing human forms of 3Rtau and 4Rtau proteins), and mtWT (homozygous transgenic mice knock-out for murine tau protein)—were analyzed following unilateral hippocampal inoculation of sarkosyl-insoluble tau fractions from the same AD and control cases. The present study reveals that (a) host tau is mandatory for tau seeding and spreading following tau inoculation from sarkosyl-insoluble fractions obtained from AD brains; (b) tau seeding does not occur following intracerebral inoculation of sarkosyl-insoluble fractions from controls; (c) tau seeding and spreading are characterized by variable genotype-dependent tau phosphorylation and tau nitration, MAP2 phosphorylation, and variable activation of kinases that co-localize with abnormal tau deposits; (d) transformation of host tau into abnormal tau is an active process associated with the activation of specific kinases; (e) tau seeding is accompanied by modifications in tau splicing, resulting in the expression of new 3Rtau and 4Rtau isoforms, thus indicating that inoculated tau seeds have the capacity to model exon 10 splicing of the host mapt or MAPT with a genotype-dependent pattern; (e) selective regional and cellular vulnerabilities, and different molecular compositions of the deposits, are dependent on the host tau of mice injected with identical AD tau inocula.

JTD Keywords: 3rtau and 4rtau, alzheimer's disease, alzheimer’s disease, brains, granulovacuolar degeneration, host tau, htau, intranuclear distribution, messenger-rna, pathological tau, propagation, protein-kinases, seeding and spreading, tauopathies, transmission, 3rtau and 4rtau, Alzheimers-disease, Alzheimer’s disease, Host tau, Htau, Seeding and spreading, Tauopathies

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

Different copolymers incorporating terpene oxide units (e.g., limonene oxide) have been evaluated considering thermal properties, degradability, and biocompatibility. Thus, polycarbonates and polyesters derived from aromatic, monocyclic and bicyclic anhydrides have been considered. Furthermore, ring substitution with myrcene terpene has been evaluated. All polymers were amorphous when evaluated directly from synthesis. However, spherulites could be observed after the slow evaporation of diluted chloroform solutions of polylimonene carbonate, with all isopropene units possessing an R configuration. This feature was surprising considering the reported information that suggested only the racemic polymer was able to crystallize. All polymers were thermally stable and showed a dependence of the maximum degradation rate temperature (from 242 °C to 342 °C) with the type of terpene oxide. The graduation of glass transition temperatures (from 44 °C to 172 °C) was also observed, being higher than those corresponding to the unsubstituted polymers. The chain stiffness of the studied polymers hindered both hydrolytic and enzymatic degradation while a higher rate was detected when an oxidative medium was assayed (e.g., weight losses around 12% after 21 days of exposure). All samples were biocompatible according to the adhesion and proliferation tests performed with fibroblast cells. Hydrophobic and mechanically consistent films (i.e., contact angles between 90° and 110°) were obtained after the evaporation of chloroform from the solutions, having different ratios of the studied biobased polyterpenes and poly(butylene succinate) (PBS). The blend films were comparable in tensile modulus and tensile strength with the pure PBS (e.g., values of 330 MPa and 7 MPa were determined for samples incorporating 30 wt.% of poly(PA-LO), the copolyester derived from limonene oxide and phthalic anhydride. Blends were degradable, biocompatible and appropriate to produce oriented-pore and random-pore scaffolds via a thermally-induced phase separation (TIPS) method and using 1,4-dioxane as solvent. The best results were attained with the blend composed of 70 wt.% PBS and 30 wt.% poly(PA-LO). In summary, the studied biobased terpene derivatives showed promising properties to be used in a blended form for biomedical applications such as scaffolds for tissue engineering.

JTD Keywords: alternating copolymerization, biobased materials, biodegradability, composites, crystallization, cyclohexene oxide, induced phase-separation, limonene oxide, mechanical-properties, polyesters, scaffolds, spherulites, terpene derivatives, thermal properties, thermally-induced phase separation, Acetone, Bio-based, Bio-based materials, Biobased materials, Biocompatibility, Biodegradability, Butenes, Cell culture, Chlorine compounds, Degradation, Evaporation, Glass transition, Limonene oxide, Monoterpenes, Phase separation, Poly (butylenes succinate), Polybutylene succinate, Property, Ring-opening copolymerization, Scaffolds, Spheru-lites, Tensile strength, Terpene derivatives, Thermal properties, Thermally induced phase separation, Thermally-induced phase separation, Thermally?induced phase separation, Thermodynamic properties, Thermogravimetric analysis

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

JTD Keywords: cancer therapy, mirnas delivery, nanocarriers, nanovesicles, neuroblastoma, pediatric cancer, quatsomes, Biodistribution, Cancer therapy, Cell engineering, Cells, Cholesterol, Controlled drug delivery, Diseases, Dna, Dysregulated ph, Lipoplex, Microrna delivery, Mirnas delivery, Nanocarriers, Nanoparticles, Nanovesicle, Nanovesicles, Neuroblastoma, Neuroblastomas, Pediatric cancer, Ph sensitive, Ph sensors, Quatsome, Quatsomes, Rna, Sirna, Sirna delivery, Sirnas delivery, Small interfering rna, Small rna, Targeted drug delivery, Tumors, Vesicles

Introduction: After a stroke, a wide range of deficits can occur with varying onset latencies. As a result, assessing impairment and recovery are enormous challenges in neurorehabilitation. Although several clinical scales are generally accepted, they are time-consuming, show high inter-rater variability, have low ecological validity, and are vulnerable to biases introduced by compensatory movements and action modifications. Alternative methods need to be developed for efficient and objective assessment. In this study, we explore the potential of computer-based body tracking systems and classification tools to estimate the motor impairment of the more affected arm in stroke patients. Methods: We present a method for estimating clinical scores from movement parameters that are extracted from kinematic data recorded during unsupervised computer-based rehabilitation sessions. We identify a number of kinematic descriptors that characterise the patients' hemiparesis (e.g., movement smoothness, work area), we implement a double-noise model and perform a multivariate regression using clinical data from 98 stroke patients who completed a total of 191 sessions with RGS. Results: Our results reveal a new digital biomarker of arm function, the Total Goal-Directed Movement (TGDM), which relates to the patients work area during the execution of goal-oriented reaching movements. The model's performance to estimate FM-UE scores reaches an accuracy of R-2: 0.38 with an error (sigma: 12.8). Next, we evaluate its reliability (r = 0.89 for test-retest), longitudinal external validity (95% true positive rate), sensitivity, and generalisation to other tasks that involve planar reaching movements (R-2: 0.39). The model achieves comparable accuracy also for the Chedoke Arm and Hand Activity Inventory (R-2: 0.40) and Barthel Index (R-2: 0.35). Conclusions: Our results highlight the clinical value of kinematic data collected during unsupervised goal-oriented motor training with the RGS combined with data science techniques, and provide new insight into factors underlying recovery and its biomarkers.

JTD Keywords: interactive feedback, motion classification, motion sensing, multivariate regression, posture monitoring, rehabilitation, stroke, Adult, Aged, Analytic method, Arm movement, Article, Barthel index, Brain hemorrhage, Cerebrovascular accident, Chedoke arm and hand activity inventory, Clinical protocol, Cognitive defect, Computer analysis, Controlled study, Convergent validity, Correlation coefficient, Disease severity, External validity, Female, Fugl meyer assessment for the upper extremity, Functional assessment, Functional status assessment, General health status assessment, Hemiparesis, Human, Interactive feedback, Ischemic stroke, Kinematics, Major clinical study, Male, Mini mental state examination, Motion classification, Motion sensing, Motor analog scale, Movement, Multivariate regression, Muscle function, Posture monitoring, Probability, Recovery, Rehabilitation, Reliability, Retrospective study, Stroke, Stroke patient, Test retest reliability, Therapy, Total goal directed movement, Upper extremities, Upper limb, Upper-limb, Wolf motor function test

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3?UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3?UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3?UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

JTD Keywords: aggregative adherence, arginine metabolism, biofilm formation, escherichia-coli, gene-expression, messenger-rna, operon, persistent diarrhea, untranslated region, Fimbria-i expression

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

JTD Keywords: exosomes, genes, lines, maintenance, pathway, proliferation, rna, stemness, tumor, Cancer

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

JTD Keywords: cell-membranes, coexistence, complex, escrt-iii, fission, guvs, lipid domains, lipid rafts, membrane fission, microcompartments, microscopy, phase separation, plasma-membrane, protein microarrays, structural basis, ternary mixtures, Escrt-iii, Giant unilamellar vesicles, Guvs, Lipid domains, Membrane fission, Microcompartments, Phase separation, Ternary mixtures

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns

Synaptic plasticity involves structural modifications in dendritic spines that are modulated by local protein synthesis and actin remodeling. Here, we investigated the molecular mechanisms that connect synaptic stimulation to these processes. We found that the phosphorylation of isoform-specific sites in eEF1A2-an essential translation elongation factor in neurons-is a key modulator of structural plasticity in dendritic spines. Expression of a nonphosphorylatable eEF1A2 mutant stimulated mRNA translation but reduced actin dynamics and spine density. By contrast, a phosphomimetic eEF1A2 mutant exhibited decreased association with F-actin and was inactive as a translation elongation factor. Activation of metabotropic glutamate receptor signaling triggered transient dissociation of eEF1A2 from its regulatory guanine exchange factor (GEF) protein in dendritic spines in a phosphorylation-dependent manner. We propose that eEF1A2 establishes a cross-talk mechanism that coordinates translation and actin dynamics during spine remodeling.

JTD Keywords: cytoskeleton, expression, f-actin, factor 1-alpha, factor 1a, messenger-rna, nucleotide exchange, protein-synthesis, synaptic plasticity, Aminoacyl-transfer-rna

Background & Aims: Prognostic models of cirrhosis underestimate disease severity for patients with cirrhosis and ascites. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein linked to hepatic neoangiogenesis and fibrogenesis. We investigated ascites MFAP4 as a predictor of transplant-free survival in patients with cirrhosis and ascites. Methods: A dual-centre observational study of patients with cirrhosis and ascites recruited consecutively in relation to a paracentesis was carried out. Patients were followed up for 1 year, until death or liver transplantation (LTx). Ascites MFAP4 was tested with the model for end-stage liver disease (MELD-Na), CLIF Consortium Acute Decompensation (CLIF-C AD), and Child-Pugh score in Cox regression models. Results: Ninety-three patients requiring paracentesis were included. Median ascites MFAP4 was 29.7 U/L [22.3–41.3], and MELD-Na was 19 [16–23]. A low MELD-Na score (<20) was observed in 49 patients (53%). During follow-up, 20 patients died (22%), and 6 received LTx (6%). High ascites MFAP4 (>29.7 U/L) was associated with 1-year transplant-free survival (p = 0.002). In Cox regression, ascites MFAP4 and MELD-Na independently predicted 1-year transplant-free survival (hazard ratio [HR] = 0.97, p = 0.03, and HR = 1.08, p = 0.01, respectively). Ascites MFAP4 and CLIF-C AD also predicted survival independently (HR = 0.96, p = 0.02, and HR = 1.05, p = 0.03, respectively), whereas only ascites MFAP4 did, controlling for the Child-Pugh score (HR = 0.97, p = 0.03, and HR = 1.18, p = 0.16, respectively). For patients with MELD-Na <20, ascites MFAP4 but not ascites protein predicted 1-year transplant-free survival (HR 0.91, p = 0.02, and HR = 0.94, p = 0.17, respectively). Conclusions: Ascites MFAP4 predicts 1-year transplant-free survival in patients with cirrhosis and ascites. In patients with low MELD-Na scores, ascites MFAP4, but not total ascites protein, significantly predicted 1-year transplant-free survival. Lay summary: Patients with cirrhosis who have fluid in the abdomen, ascites, are at an increased risk of death and in need for liver transplantation. Our study identified patients with ascites and a poor prognosis by measuring microfibrillar associated protein 4 (MFAP4), a protein present in the abdominal fluid. Patients with low levels of the MFAP4 protein are at particularly increased risk of death or liver transplantation, suggesting that clinical care should be intensified in this group of patients. © 2021 The Authors

JTD Keywords: biomarker, clif-c ad, clif consortium acute decompensation, cps, child-pugh score, crp, c-reactive protein, ct, computed tomography, decompensated, ecm, extracellular matrix, fibrosis, fluid protein, gfr, glomerular filtration rate, hr, hazard ratio, inr, internationalised normal ratio, liver disease, liver-cirrhosis, ltx, liver transplantation, markers, meld-na, model for end-stage liver disease, mfap4, microfibrillar associated protein 4, mortality, nash, non-alcoholic steatohepatitis, natural-history, prognosis, risk-factors, sbp, spontaneous bacterial peritonitis, scores, stage, Biomarker, Decompensated, Egfr, estimated gfr, Fibrosis, Liver disease, Mortality, Prognosis, Spontaneous bacterial peritonitis

Significance: The incidence of chronic wounds is increasing due to our aging population and the augment of people afflicted with diabetes. With the extended knowledge on the biological mechanisms underlying these diseases, there is a novel influx of medical technologies into the conventional wound care market. Recent Advances: Several nanotechnologies have been developed demonstrating unique characteristics that address specific problems related to wound repair mechanisms. In this review, we focus on the most recently developed nanotechnology-based therapeutic agents and evaluate the efficacy of each treatment in in vivo diabetic models of chronic wound healing. Critical Issues: Despite the development of potential biomaterials and nanotechnology-based applications for wound healing, this scientific knowledge is not translated into an increase of commercially available wound healing products containing nanomaterials. Future Directions: Further studies are critical to provide insights into how scientific evidences from nanotechnology-based therapies can be applied in the clinical setting.

JTD Keywords: chronic, diabetes, liposomes, nanofibers, nanoparticles, Chronic, Chronic wound, Diabetes, Diabetic wound, Diabetic-rats, Dressings, Drug mechanism, Extracellular-matrix, Growth-factor, Human, In-vitro, Liposome, Liposomes, Mesenchymal stem-cells, Metal nanoparticle, Nanofiber, Nanofibers, Nanofibrous scaffolds, Nanoparticles, Nanotechnology, Nonhuman, Polyester, Polymer, Polysaccharide, Priority journal, Protein, Review, Self assembled protein nanoparticle, Silk fibroin, Skin wounds, Wound healing, Wound healing promoting agent

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3?, whose activity is inhibited by the cellular prion protein (PrPC), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrPC and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrPC and the implication of GSK3? in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrPC ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrPC expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrPC levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3? activity downstream from PrPC in this phenomenon. Our results indicate that PrPC plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3?. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: alternative splicing, alzheimer's disease, alzheimers-disease, alzheimer’s disease, amyloid-beta, cellular prion protein, frontotemporal dementia, glycogen-synthase kinase-3, gsk3 beta, gsk3?, gsk3β, messenger-rna, microtubule-associated protein tau, neurofibrillary tangles, progressive supranuclear palsy, promotes neuronal differentiation, stem-cells, tauopathies, Alternative splicing, Alzheimer’s disease, Cellular prion protein, Gsk3?, Microtubule-associated protein tau, Tauopathies

© 2020 Elsevier Ltd Protein phase transitions are particularly amenable for cell signalling as these highly cooperative processes allow cells to make binary decisions in response to relatively small intracellular changes. The different processes of condensate formation and the distinct material properties of the resulting condensates provide a dictionary to modulate a range of decisions on cell fate. We argue that, on the one hand, the reversibility of liquid demixing offers a chance to arrest cell growth under specific circumstances. On the other hand, the transition to amyloids is better suited for terminal decisions such as those leading to apoptosis and necrosis. Here, we review recent examples of both scenarios, highlighting how mutations in signalling proteins affect the formation of biomolecular condensates with drastic effects on cell survival.

JTD Keywords: amyloid, cell death, deep mutagenesis, llps, rna-binding proteins, Amyloid, Cell death, Deep mutagenesis, Llps, Phase transition, Proteins, Rna-binding proteins, Signal transduction

Background & Aims: MicroRNAs (miRNAs) circulate in several body fluids and can be useful biomarkers. The aim of this study was to identify blood-circulating miRNAs associated with cirrhosis progression and acute-on-chronic liver failure (ACLF). Methods: Using high-throughput screening of 754 miRNAs, serum samples from 45 patients with compensated cirrhosis, decompensated cirrhosis, or ACLF were compared with those from healthy individuals (n = 15). miRNA levels were correlated with clinical parameters, organ failure, and disease progression and outcome. Dysregulated miRNAs were evaluated in portal and hepatic vein samples (n = 33), liver tissues (n = 17), and peripheral blood mononuclear cells (PBMCs) (n = 16). Results: miRNA screening analysis revealed that circulating miRNAs are dysregulated in cirrhosis progression, with 51 miRNAs being differentially expressed among all groups of patients. Unsupervised clustering and principal component analysis indicated that the main differences in miRNA expression occurred at decompensation, showing similar levels in patients with decompensated cirrhosis and those with ACLF. Of 43 selected miRNAs examined for differences among groups, 10 were differentially expressed according to disease progression. Moreover, 20 circulating miRNAs were correlated with model for end-stage liver disease and Child-Pugh scores. Notably, 11 dysregulated miRNAs were associated with kidney or liver failure, encephalopathy, bacterial infection, and poor outcomes. The most severely dysregulated miRNAs (i.e. miR-146a5p, miR-26a-5p, and miR-191-5p) were further evaluated in portal and hepatic vein blood and liver tissue, but showed no differences. However, PBMCs from patients with cirrhosis showed significant downregulation of miR-26 and miR-146a, suggesting a extrahepatic origin of some circulating miRNAs. Conclusions: This study is a repository of circulating miRNA data following cirrhosis progression and ACLF. Circulating miRNAs were profoundly dysregulated during the progression of chronic liver disease, were associated with failure of several organs and could have prognostic utility. Lay summary: Circulating miRNAs are small molecules in the blood that can be used to identify or predict a clinical condition. Our study aimed to identify miRNAs for use as biomarkers in patients with cirrhosis or acute-on-chronic liver failure. Several miRNAs were found to be dysregulated during the progression of disease, and some were also related to organ failure and disease-related outcomes. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL).

JTD Keywords: aclf, acute-on-chronic liver failure, alt, alanine aminotransferase, ast, aspartate aminotransferase, biomarkers, chronic liver disease, cxcl10, c-x-c motif chemokine ligand 10, ef clif, european foundation for the study of chronic liver failure, foxo, forkhead box o, inr, international normalised ratio, ldh, lactate dehydrogenase, liver decompensation, mapk, mitogen-activated protein kinase, meld, model for end-stage liver disease, nash, non-alcoholic steatohepatitis, non-coding rnas, pbmcs, peripheral blood mononuclear cells, pca, principal component analysis, tgf, transforming growth factor, tips, transjugular intrahepatic portosystemic shunt, Biomarkers, Chronic liver disease, Expression, Liver decompensation, Markers, Mir-146a, Non-coding rnas, Qpcr, quantitative pcr

The puropse of this study was to evaluate associations of cisterna chyli (CCh) diameter with portal hemodynamics and the influence of TIPS-creation in cirrhotic patients. 93 cirrhotic patients (57 male, mean age 59 years) received CT prior to TIPS-creation. 38/93 additionally underwent post-interventional CT. CCh-diameter was measured. After categorization into patients with and without large venous collaterals (i.e. > 6 mm), data were analyzed regarding associations between CCh-diameter, clinical and portal-hemodynamic parameters and diameter-changes after TIPS-creation. Patient survival post-TIPS was analyzed. Median portosystemic pressure-gradient decreased from 20 to 9 mmHg after TIPS-creation. Large venous collaterals were observed in 59 patients. In 69/93 patients (74.2%) the CCh was detectable. Mean pre-interventional diameter was 9.4 ± 2.7 mm (large collaterals: 8.7 ± 2.0 mm, no large collaterals: 10.7 ± 3.2 mm, p = 0.003). CCh-diameter correlated strongly with pre-TIPS portal-pressure (Rs = 0.685, p = 0.0001), moderately with portosystemic-gradient (Rs = 0.524, p = 0.006), liver shear-wave-elastography (Rs = 0.597, p = 0.004) and spleen size (Rs = 0.501, p = 0.01) in patients without large collaterals, but not in patients with large collaterals. Post-TIPS CCh-diameter decreased significantly from 10.2 ± 2.8 mm to 8.3 ± 3.0 mm (p < 0.001). Patients without a detectable CCh on CT survived significantly shorter. The diameter of the CCh is associated with portal-pressure and decreases after TIPS-creation in cirrhotic patients, reflecting a portal decompression mechanism via the lymphatic system. Lack of larger central lymphatics detectable on CT may be associated with shorter survival.

JTD Keywords: circulation, cisterna chyli, fluid, hepatic cirrhosis, shear-wave elastography, thoracic-duct, vessels, Significant portal-hypertension

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This study aims to investigate noninvasive indices of neuromechanical coupling (NMC) and mechanical efficiency (MEff) of parasternal intercostal muscles. Gold standard assessment of diaphragm NMC requires using invasive techniques, limiting the utility of this procedure. Nonin-vasive NMC indices of parasternal intercostal muscles can be calculated using surface mechano-myography (sMMGpara) and electromyography (sEMGpara). However, the use of sMMGpara as an in-spiratory muscle mechanical output measure, and the relationships between sMMGpara, sEMGpara, and simultaneous invasive and noninvasive pressure measurements have not previously been eval-uated. sEMGpara, sMMGpara, and both invasive and noninvasive measurements of pressures were recorded in twelve healthy subjects during an inspiratory loading protocol. The ratios of sMMGpara to sEMGpara, which provided muscle-specific noninvasive NMC indices of parasternal intercostal muscles, showed nonsignificant changes with increasing load, since the relationships between sMMGpara and sEMGpara were linear (R2 = 0.85 (0.75–0.9)). The ratios of mouth pressure (Pmo) to sEMGpara and sMMGpara were also proposed as noninvasive indices of parasternal intercostal muscle NMC and MEff, respectively. These indices, similar to the analogous indices calculated using invasive transdiaphragmatic and esophageal pressures, showed nonsignificant changes during threshold loading, since the relationships between Pmo and both sEMGpara (R2 = 0.84 (0.77–0.93)) and sMMGpara (R2 = 0.89 (0.85–0.91)) were linear. The proposed noninvasive NMC and MEff indices of parasternal intercostal muscles may be of potential clinical value, particularly for the regular assessment of patients with disordered respiratory mechanics using noninvasive wearable and wireless devices.

JTD Keywords: inspiratory threshold loading, neuromechanical coupling, parasternal intercostal muscles, respiratory pressure, surface electromyography, surface mechanomyography, Inspiratory threshold loading, Neuromechanical coupling, Parasternal intercostal mus-cles, Respiratory pressure, Surface electromyography, Surface mechanomyography

© 2020 Elsevier B.V. We examine different approaches for the controlled release of L-lactate, which is a signaling molecule that participates in tissue remodeling and regeneration, such as cardiac and muscle tissue. Robust, flexible, and self-supported 3-layers films made of two spin-coated poly(lactic acid) (PLA) layers separated by an electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT) layer, are used as loading and delivery systems. Films with outer layers prepared using homochiral PLA and with nanoperforations of diameter 146 ± 70 experience more bulk erosion, which also contributes to the release of L-lactic acid, than those obtained using heterochiral PLA and with nanoperforations of diameter 66 ± 24. Moreover, the release of L-lactic acid as degradation product is accelerated by applying biphasic electrical pulses. The four approaches used for loading extra L-lactate in the 3-layered films were: incorporation of L-lactate at the intermediate PEDOT layer as primary dopant agent using (1) organic or (2) basic water solutions as reaction media; (3) substitution at the PEDOT layer of the ClO4− dopant by L-lactate using de-doping and re-doping processes; and (4) loading of L-lactate at the outer PLA layers during the spin-coating process. Electrical stimuli were applied considering biphasic voltage pulses and constant voltages (both negative and positive). Results indicate that the approach used to load the L-lactate has a very significant influence in the release regulation process, affecting the concentration of released L-lactate up to two orders of magnitude. Among the tested approaches, the one based on the utilization of the outer layers for loading, approach (4), can be proposed for situations requiring prolonged and sustained L-lactate release over time. The biocompatibility and suitability of the engineered films for cardiac tissue engineering has also been confirmed using cardiac cells.

JTD Keywords: biphasic voltage pulse, cardiac tissue regeneration, cardiomyocytes proliferation, conducting polymer, nanoperforated films, sustained delivery, Biphasic voltage pulse, Cardiac tissue regeneration, Cardiomyocytes proliferation, Conducting polymer, Nanoperforated films, Sustained delivery

© 2020 Elsevier Inc. Using single-cell RNA sequencing, Susztak and colleagues, show major changes in cell diversity in mouse models of kidney fibrosis. Proximal tubule (PT) cells are highly vulnerable to dysfunction in fibrosis and show altered differentiation. Nuclear receptors such as ESRRA maintain both PT cell metabolism and differentiation by directly regulating PT-cell-specific genes.

JTD Keywords: chronic kidney disease, esrra, fatty-acid oxidation, fibrosis, kidney, organoids, ppara, proximal tubule cells, single-cell atac sequencing, Chronic kidney disease, Esrra, Fatty-acid oxidation, Fibrosis, Kidney, Organoids, Ppara, Proximal tubule cells, Single-cell atac sequencing, Single-cell rna sequencing

Goel and colleagues show that CDK4/6 inhibition induces global chromatin changes mediated by AP-1 factors, which mediate key biological and clinical effects in breast cancer. Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that are enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several activator protein-1 transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.

JTD Keywords: Abemaciclib, Androgen receptor, Animal experiment, Animal model, Animal tissue, Apoptosis, Article, Breast cancer, C-jun, Cancer cell, Carcinoembryonic antigen related cell adhesion molecule 1, Caspase 3, Cell cycle arrest, Cells, Chromatin, Chromatin immunoprecipitation, Controlled study, Cyclin dependent kinase 4, Cyclin dependent kinase 6, Dna damage, Epidermal growth factor receptor 2, Estrogen receptor, Female, Flow cytometry, Fulvestrant, Hla drb1 antigen, Human, Human cell, Immunoblotting, Immunogenicity, Immunoprecipitation, Interferon, Luciferase assay, Mcf-7 cell line, Mda-mb-231 cell line, Microarray analysis, Morphogenesis, Mouse, Nonhuman, Palbociclib, Protein, Protein expression, Rb, Resistance, Rna polymerase ii, Rna sequence, Selective-inhibition, Senescence, Short tandem repeat, Signal transduction, Tamoxifen, Transcription elongation, Transcription factor, Transcription factor ap 1, Transcriptome, Tumor biopsy, Tumor differentiation, Tumor spheroid, Tumor xenograft, Vinculin, Whole exome sequencing

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell–cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell–substrate adhesion in the tissue engineering strategies for cartilage repair.

JTD Keywords: Dendrimer, Nanopatterning, RGD, Mesenchymal cell condensation, Cell–cell interactions, YAP, Chondrogenesis

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

The electro-chemo-mechanical response of robust and flexible free-standing films made of three nanoperforated poly(lactic acid) (pPLA) layers separated by two anodically polymerized poly(3,4-ethylenedioxythiophene) (PEDOT) layers has been demonstrated. The mechanical and electrochemical properties of these films, which are provided by pPLA and PEDOT, respectively, have been studied by nanoindentation, cyclic voltammetry, and galvanostatic charge-discharge assays. The unprecedented combination of properties obtained for this system is appropriated for its utilization as a Faradaic motor, also named artificial muscle. Application of square potential waves has shown important bending movements in the films, which can be repeated for more than 500 cycles without damaging its mechanical integrity. Furthermore, the actuator is able to push a huge amount of mass, as it has been proved by increasing the mass of the passive pPLA up to 328% while keeping the mass of electroactive PEDOT unaltered.

JTD Keywords: Actuator, Artificial muscle, Conducting polymer, Nanoindentation

Ranging from miniaturized biological robots to organoids, multi-cellular engineered living systems (M-CELS) pose complex ethical and societal challenges. Some of these challenges, such as how to best distribute risks and benefits, are likely to arise in the development of any new technology. Other challenges arise specifically because of the particular characteristics of M-CELS. For example, as an engineered living system becomes increasingly complex, it may provoke societal debate about its moral considerability, perhaps necessitating protection from harm or recognition of positive moral and legal rights, particularly if derived from cells of human origin. The use of emergence-based principles in M-CELS development may also create unique challenges, making the technology difficult to fully control or predict in the laboratory as well as in applied medical or environmental settings. In response to these challenges, we argue that the M-CELS community has an obligation to systematically address the ethical and societal aspects of research and to seek input from and accountability to a broad range of stakeholders and publics. As a newly developing field, M-CELS has a significant opportunity to integrate ethically responsible norms and standards into its research and development practices from the start. With the aim of seizing this opportunity, we identify two general kinds of salient ethical issues arising from M-CELS research, and then present a set of commitments to and strategies for addressing these issues. If adopted, these commitments and strategies would help define M-CELS as not only an innovative field, but also as a model for responsible research and engineering.

JTD Keywords: Ethics, Society, Governance, Emergence, Moral considerability, Responsible innovation

Background: Severe hepatic steatosis shows a high prevalence and contributes to morbidity and mortality in human immunodeficiency virus (HIV) infected patients. Known risk factors include obesity, dyslipidaemia and features of metabolic syndrome. Fibroblast growth factor 21 (FGF-21) is involved with hepatic glucose and lipid metabolism. This study aimed to evaluate FGF-21 as a biomarker for severe hepatic steatosis in non-obese HIV-infected patients. Methods: This is a prospective, cross-sectional, monocentric study including HIV-infected out-patients. Hepatic steatosis was measured via controlled attenuation parameter (CAP) using FibroScan 502 touch (ECHOSENS, France). Severe hepatic steatosis was defined at CAP ≥ 253 dB/m. Peripheral blood samples were collected and plasma was analysed for FGF-21. Demographic and clinical characteristics were collected from patient's health records. Results: In total, 73 non-obese HIV-monoinfected patients were included in this study. Prevalence of severe hepatic steatosis was 41%. Patients with severe hepatic steatosis showed significantly higher levels of FGF-21. Univariate analysis revealed FGF-21, BMI, hyperlipidaemia, ALT levels and arterial hypertension as significant, while multivariate analysis showed only FGF-21, arterial hypertension and ALT levels as significant independent risk factors for severe hepatic steatosis. Conclusion: This study presents FGF-21 as an independent and stronger predictor of severe hepatic steatosis than blood lipids in HIV-infected patients. Moreover, arterial hypertension and ALT levels predict severe steatosis even in non-obese HIV-monoinfected patients. Furthermore, this study supports existing metabolic risk factors and expands them to non-obese HIV-infected patients.

JTD Keywords: BMI, CAP, Dyslipidaemia, FGF-21, Fibroscan, HIV, Hyperlipidaemia, Liver, NAFLD, NASH, Steatosis

Our understanding of body ownership largely relies on the so-called Rubber Hand Illusion (RHI). In this paradigm, synchronous stroking of the real and the rubber hands leads to an illusion of ownership of the rubber hand provided that it is physically, anatomically, and spatially plausible. Self-attribution of an artificial hand also occurs during visuomotor synchrony. In particular, participants experience ownership over a virtual or a rubber hand when the visual feedback of self-initiated movements follows the trajectory of the instantiated motor commands, such as in the Virtual Hand Illusion (VHI) or the moving Rubber Hand Illusion (mRHI). Evidence yields that both when the cues are triggered externally (RHI) and when they result from voluntary actions (VHI and mRHI), the experience of ownership is established through bottom-up integration and top-down prediction of proximodistal cues (visuotactile or visuomotor) within the peripersonal space. It seems, however, that depending on whether the sensory signals are externally (RHI) or self-generated (VHI and mRHI), the top-down expectation signals are qualitatively different. On the one hand, in the RHI the sensory correlations are modulated by top-down influences which constitute empirically induced priors related to the internal (generative) model of the body. On the other hand, in the VHI and mRHI body ownership is actively shaped by processes which allow for continuous comparison between the expected and the actual sensory consequences of the actions. Ample research demonstrates that the differential processing of the predicted and the reafferent information is addressed by the central nervous system via an internal (forward) model or corollary discharge. Indeed, results from the VHI and mRHI suggest that, in action-contexts, the mechanism underlying body ownership could be similar to the forward model. Crucially, forward models integrate across all self-generated sensory signals including not only proximodistal (i.e., visuotactile or visuomotor) but also purely distal sensory cues (i.e., visuoauditory). Thus, if body ownership results from a consistency of a forward model, it will be affected by the (in)congruency of purely distal cues provided that they inform about action-consequences and are relevant to a goal-oriented task. Specifically, they constitute a corrective error signal. Here, we explicitly addressed this question. To test our hypothesis, we devised an embodied virtual reality-based motor task where action outcomes were signaled by distinct auditory cues. By manipulating the cues with respect to their spatial, temporal and semantic congruency, we show that purely distal (visuoauditory) feedback which violates predictions about action outcomes compromises both performance and body ownership. These results demonstrate, for the first time, that body ownership is influenced by not only externally and self-generated cues which pertain to the body within the peripersonal space but also those arising outside of the body. Hence, during goal-oriented tasks body ownership may result from the consistency of forward models.

JTD Keywords: Body ownership, Internal forward model, Goal-oriented behavior, Multisensory integration, Top-down prediction

Introduction: Vulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident-intruder confrontations to study the brain corticostriatal-function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB1 receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures. Aims and methods: The investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid-like molecules and changes in the transcriptome. Results: Stressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N-arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB1 activation, N-oleoylethanolamide and Npalmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co-administration with both cannabinoids induced an up-regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2-arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co-administration tended to produce a null effect under stress. Conclusions: The endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha-6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid-like mediators might be a valuable option for treating stress-related neuropsychiatric symptoms

JTD Keywords: CB1, Chrna6 and Slc6a4, Fkbp5, Pychosocial stress

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

JTD Keywords: Bioengineering, Dendrimer, Nanopattern, Arginine-Glycine-Aspartic Acid (RGD), Atomic Force Microscopy (AFM), Cell Adhesion, Mesenchymal Stem Cells (Mscs), Chondrogenesis

Although discriminating self from nonself is a cardinal animal trait, metazoan allorecognition genes do not appear to be homologous. Here, we characterize the Aggregation Factor (AF) gene family, which encodes putative allorecognition factors in the demosponge Amphimedon queenslandica, and trace its evolution across 24 sponge (Porifera) species. The AF locus in Amphimedon is comprised of a cluster of five similar genes that encode Calx-beta and Von Willebrand domains and a newly defined Wreath domain, and are highly polymorphic. Further AF variance appears to be generated through individualistic patterns of RNA editing. The AF gene family varies between poriferans, with protein sequences and domains diagnostic of the AF family being present in Amphimedon and other demosponges, but absent from other sponge classes. Within the demosponges, AFs vary widely with no two species having the same AF repertoire or domain organization. The evolution of AFs suggests that their diversification occurs via high allelism, and the continual and rapid gain, loss and shuffling of domains over evolutionary time. Given the marked differences in metazoan allorecognition genes, we propose the rapid evolution of AFs in sponges provides a model for understanding the extensive diversification of self-nonself recognition systems in the animal kingdom.

JTD Keywords: Aggregation factor, Allorecognition, Intron phase, Polymorphism, Porifera, RNA editing

Background: Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils. Results: To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator. Conclusion: This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.

JTD Keywords: CRP, Entner-Doudoroff pathway, Nutrient starvation, Rhodococcus, RNA-Seq, Triacylglycerol

Prolonged and sustained stimulation of the hypothalamo-pituitary-adrenal axis have adverse effects on numerous brain regions, including the cerebellum. Motor coordination and motor learning are essential for animal and require the regulation of cerebellar neurons. The G-protein-coupled cannabinoid CB1 receptor coordinates synaptic transmission throughout the CNS and is of highest abundance in the cerebellum. Accordingly, the aim of this study was to investigate the long-lasting effects of chronic psychosocial stress on motor coordination and motor learning, CB1 receptor expression, endogenous cannabinoid ligands and gene expression in the cerebellum. After chronic psychosocial stress, motor coordination and motor learning were impaired as indicated the righting reflex and the rota-rod. The amount of the endocannabinoid 2-AG increased while CB1 mRNA and protein expression were downregulated after chronic stress. Transcriptome analysis revealed 319 genes differentially expressed by chronic psychosocial stress in the cerebellum; mainly involved in synaptic transmission, transmission of nerve impulse, and cell-cell signaling. Calreticulin was validated as a stress candidate gene. The present study provides evidence that chronic stress activates calreticulin and might be one of the pathological mechanisms underlying the motor coordination and motor learning dysfunctions seen in social defeat mice.

JTD Keywords: Psychosocial stress, Cerebellum, Calreticulin, Endocannabinoid system, Behavior, RNA seq.

Summary: Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

JTD Keywords: Haemophilia A, Leucocytes, RNA splicing, Splice site mutation, Synonymous mutation

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ~ 2–4), we found that the DNA dielectric constant is ~ 8, considerably higher than the value of ~ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

JTD Keywords: Atomic force microscopy, Atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, capsid protein, DNA, double stranded DNA, amino acid composition, article, atomic force microscopy, bacteriophage, bacteriophage T7, dielectric constant, dipole, DNA binding, DNA packaging, DNA structure, electron microscopy, ligand binding, nonhuman, polarization, priority journal, protein analysis, protein DNA interaction, scanning probe microscopy, static electricity, virion, virus capsid, virus particle, atomic force microscopy, atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, Bacteriophage T7, Capsid, Cations, Dielectric Spectroscopy, DNA, DNA, Viral, DNA-Binding Proteins, Electrochemical Techniques, Ligands, Microscopy, Atomic Force, Models, Chemical, Nuclear Proteins

Study Objectives: To test the hypotheses that brain oxygen partial pressure (PtO2) in response to obstructive apneas changes with age and that it might lead to different levels of cerebral tissue oxidative stress. Design: Prospective controlled animal study. Setting: University laboratory. Participants: Sixty-four male Wistar rats: 32 young (3 mo old) and 32 aged (18 mo). Interventions: Protocol 1: Twenty-four animals were subjected to obstructive apneas (50 apneas/h, lasting 15 sec each) or to sham procedure for 50 min. Protocol 2: Forty rats were subjected to obstructive apneas or sham procedure for 4 h. Measurements and Results: Protocol 1: Real-time PtO2 measurements were performed using a fast-response oxygen microelectrode. During successive apneas cerebral cortex PtO2 presented a different pattern in the two age groups; there was a fast increase in young rats, whereas it remained without significant changes between the beginning and the end of the protocol in the aged group. Protocol 2: Brain oxidative stress assessed by lipid peroxidation increased after apneas in young rats (1.34 ± 0.17 nmol/mg of protein) compared to old ones (0.63 ± 0.03 nmol/mg), where a higher expression of antioxidant enzymes was observed. Conclusions: The results suggest that brain oxidative stress in aged rats is lower than in young rats in response to recurrent apneas, mimicking obstructive sleep apnea. This could be due to the different PtO2 response observed between age groups and the increased antioxidant expression in aged rats.

JTD Keywords: Aging, Animal model, Obstructive apnea, Oxidative stress, Tissue oxygenation, antioxidant, glutathione disulfide, aged, animal experiment, animal model, animal tissue, apnea, arterial oxygen saturation, article, brain cortex, brain oxygen tension, brain tissue, controlled study, groups by age, hypoxia, lipid peroxidation, male, nonhuman, oxidative stress, pressure, priority journal, rat

Understanding how charges move through and between biomolecules is a fundamental question that constitutes the basis for many biological processes. On the other hand, it has potential applications in the design of sensors based on biomolecules and single molecule devices. In this review we introduce the study of the electron transfer (ET) process in biomolecules, providing an overview of the fundamental theory behind it and the different experimental approaches. The ET in proteins is introduced by reviewing a complete electronic characterization of a redox protein (azurin) using electrochemical scanning tunnelling microscopy (ECSTM). The ET process in DNA is overviewed and results from different experimental approaches are discussed. Finally, future directions in the study of the ET process in biomolecules are introduced as well as examples of possible technological applications.

JTD Keywords: Bioelectrochemistry, Biomolecular electronics, Charge transfer, Nanobiodevice, Single-molecule junction

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1. kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7. kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6. kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8. kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

JTD Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering

Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (RL) and elastance (EL) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

JTD Keywords: Lung bioengineering, Lung decellularization, Organ scaffold, dodecyl sulfate sodium, animal tissue, article, artificial ventilation, compliance (physical), controlled study, enzyme chemistry, extracellular matrix, female, flow, lung, lung decellularization, lung pressure, lung resistance, mouse, nonhuman, positive end expiratory pressure, priority journal, rigidity, tissue engineering, trachea pressure

DNA testing requires the development of sensitive and fast devices to measure the presence of nucleic acid sequences by DNA hybridization. In this paper, a simple and label-free DNA-biosensor has been investigated based on the detection of DNA hybridization on layered double hydroxide (LDH) nanomaterials with special emphasis on targeting long single stranded DNA sequences. First, the immobilization of a 20 bases long DNA probe on a thin layer of Mg2AlCO3 and Mg3AlCO3 LDH was studied. Then, DNA hybridization reaction was detected by means of Electrochemical Impedance Spectroscopy. The resulting biosensor showed a high sensitivity for the detection of 80 bases long DNA complementary sequences. The dynamic range was 18–270 ng/ml with a detection limit lower than 1.8 ng/ml.

JTD Keywords: DNA-biosensor, Nanomaterials, Layered double hydroxide, Self-assembly

The potential use of a micromachined thermopile based sensor device for analyzing natural gas is explored. The sensor consists of a thermally isolated hotplate which is heated by the application of a sequence of programmed voltages to an integrated heater. Once the hotplate reaches a stationary temperature, the thermopile provides a signal proportional to the hotplate temperature. These signals are processed in order to determine different natural gas properties. Sensor response is mainly dependent on the thermal conductivity of the surrounding gas at different temperatures. Seven predicted properties (normal density, Superior Heating Value, Wobbe index and the concentrations of methane, ethane, carbon dioxide and nitrogen) are calibrated against sensor signals by using multivariate regression, in particular Partial Least Squares. Experimental data have been used for calibration and validation. Results show property prediction capability with reasonable accuracy except for prediction of carbon dioxide concentration. A detailed uncertainty analysis is provided to better understand the metrological limits of the system. These results imply for the first time the possibility of designing unprecedented low-cost natural gas analyzers. The concept may be extended to other constrained gas mixtures (e.g. of a known number of components) to enable low-cost multicomponent gas analyzers.

JTD Keywords: Gas sensor, Natural gas, MEMS, Superior Heating Value, density, PLS

This article reports on the research and development of a cutting-edge biomedical device for continuous in-vivo glucose monitoring. This entirely public-funded process of technological innovation has been conducted at the University of Barcelona within a context of converging technologies involving the fields of medicine, physics, chemistry, biology, telecommunications, electronics and energy. The authors examine the value chain and the market challenges faced by in-vivo implantable biomedical devices based on nanotechnologies. In so doing, they trace the process from the point of applied research to the final integration and commercialization of the product, when the social rate of return from academic research can be estimated. Using a case-study approach, the paper also examines the high-tech activities involved in the development of this nano-enabled device and describes the technology and innovation management process within the value chain conducted in a University-Hospital-Industry-Administration-Citizens framework. Here, nanotechnology is seen to represent a new industrial revolution, boosting the biomedical devices market. Nanosensors may well provide the tools required for investigating biological processes at the cellular level in vivo when embedded into medical devices of small dimensions, using biocompatible materials, and requiring reliable and targeted biosensors, high speed data transfer, safely stored data, and even energy autonomy.

JTD Keywords: Biomedical device, Diabetes, Innovation management, Nanobiosensor, Nanotechnology, Research commercialization, Technology transfer, Academic research, Applied research, Barcelona, Biocompatible materials, Biological process, Biomedical analysis, Biomedical devices, Cellular levels, Converging technologies, Glucose monitoring, High-speed data transfer, Implantable biomedical devices, Implantable devices, In-vivo, Industrial revolutions, Innovation management, Medical Devices, Nanobiosensor, Rate of return, Research and development, Technological innovation, Value chains, Biological materials, Biomedical engineering, Biosensors, Commerce, Data transfer, Earnings, Engineering education, Glucose, Implants (surgical), Industrial research, Innovation, Medical problems, Nanosensors, Nanotechnology, Technology transfer, Equipment

This paper presents a novel teleoperation paradigm, the Virtual Robot (VR), focused on facilitating the surgeon tasks in minimally invasive robotic surgery. The VR has been conceived to increase the range of applicability of traditional master slave teleoperation architectures by means of an automatic cooperative behavior that assigns the execution of the ongoing task to the most suitable robot. From the user's point of view, the VR internal operation must be automatic and transparent. A set of evaluation indexes have been developed to obtain the suitability of each robot as well as an algorithm to determine the optimal instant of time to execute a task transfer. Several experiments demonstrate the usefulness of the VR, as well as indicates the next steps of the research.

JTD Keywords: Cameras, Collision avoidance, Indexes, Joints, Robots, Surgery, Trajectory, Medical robotics, Surgery, Telerobotics, VR internal operation, Automatic cooperative behavior, Evaluation indexes, Master slave teleoperation architectures, Minimally invasive robotic surgery, Task transfer, Virtual robot

During alternating ventilation (AV) one lung is inflating while the other is deflating. Considering the possible respiratory and hemodynamic advantages of AV, we investigated its effects during increased intra-abdominal pressure (IAP = 10 mmHg). In Sprague-Dawley rats (n = 6, 270–375 g) the main bronchi were independently cannulated, and respiratory mechanics determined while animals underwent different ventilatory patterns: synchronic ventilation without increased IAP (SV-0), elevated IAP during SV (SV-10), and AV with elevated IAP (AV-10). Thirty-three other animals (SV-0, n = 10; SV-10, n = 11 and AV-10, n = 12) were ventilated during 3 h. Mean arterial pressure (MAP), and lung histology were assessed. Increased IAP resulted in significantly higher elastances (p < 0.001), being AV-10 lower than SV-10 (p < 0.020). SV-10 showed higher central venous pressure (p < 0.003) than S-0; no change was observed in AV-10. Wet/dry lung weight ratio was lower in AV-10 than SV-10 (p = 0.009). Application of AV reduced hemodynamic and lung impairments induced by increased IAP during SV.

JTD Keywords: Alternating ventilation, Respiratory mechanics, Intra-abdominal pressure, Hemodynamic, Mechanical ventilation, Animal model

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.

JTD Keywords: Force spectroscopy, Atomic force microscopy, Lipid bilayer, Nanomechanics

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

JTD Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

The H-NS protein plays a significant role in the modulation of gene expression in Gram-negative bacteria. Whereas isolation and characterization of hns mutants in Escherichia coli, Salmonella and Shigella represented critical steps to gain insight into the modulatory role of H-NS, it has hitherto not been possible to isolate hns mutants in Yersinia. The hns mutation is considered to be deleterious in this genus. To study the modulatory role of H-NS in Yersinia we circumvented hns lethality by expressing in Y. enterocolitica a truncated H-NS protein known to exhibit anti-H-NS activity in E. coli (H-NST(EPEC)). Y. enterocolitica cells expressing H-NST(EPEC) showed an altered growth rate and several differences in the protein expression pattern, including the ProV protein, which is modulated by H-NS in other enteric bacteria. To further confirm that H-NST(EPEC) expression in Yersinia can be used to demonstrate H-NS-dependent regulation in this genus, we used this approach to show that H-NS modulates expression of the YmoA protein.

JTD Keywords: Bacterial Proteins/biosynthesis/genetics/ physiology, DNA-Binding Proteins/biosynthesis/genetics/ physiology, Electrophoresis, Gel, Two-Dimensional, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Essential, Proteome/analysis, RNA, Bacterial/biosynthesis, RNA, Messenger/biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Sequence Deletion, Yersinia enterocolitica/chemistry/genetics/growth & development/ physiology