Publications

Background/Objectives: The question addressed in the current work is to develop a simulation of a pharmaceutical process (DNA encapsulation within lipid nanoparticles using a microfluidic micromixer) which will be of utility to the end users (laboratory-scale formulation development). The simulation and the microfluidic approach also address sustainability issues, such as reducing the environmental impact of the process itself, and reducing the need for physical testing. The paper details the implementation and validation, taking into account key performance indicators and control parameters. Methods: The main method applied for simulation development is a novel multi-agent approach to incorporate stochastic probabilistic behavior, combined with theoretical definitions from the process experts and relevant literature, and data/results from laboratory-scale experiments with different parameter configurations. Results: The simulation was implemented as a representation of the real physical process, reproducing the relationships between process parameters (flow rates) and experimental key performance indicators (capsule diameter, poly dispersion index, encapsulation efficiency). The simulation results demonstrated a general agreement with the empirical results and provided useful predictive insights for the laboratory experiments. Conclusions: The simulation has potential as a support tool for laboratory experiments to reduce physical testing and indicate the most promising configurations on which to focus, with potential savings in time, resources and other costs.

JTD Keywords: Calibration, Encapsulation, Lipid nanoparticles, Microfluidics, Micromixer, Multi-agents, Optimization, Process simulation, Sustainability

Highlights What are the main findings? center dot The smartphone-based portable monitoring system enabled detection of previously undiagnosed sleep apnea among post-stroke patients undergoing rehabilitation. center dot Greater sleep-disordered respiratory events and nocturnal hypoxemia were associated with worse baseline disability and lower rehabilitation metrics. What are the implications of the main findings? center dot The portable system was easy to use, facilitating sleep apnea detection after stroke and supporting broader implementation in rehabilitation settings. center dot Routine screening for sleep-disordered breathing at admission may enable earlier diagnosis and management in patients with substantial hypoxemia/event burden that could slow functional recovery. Sleep-disordered breathing (SDB) is common after stroke and may negatively influence recovery, yet it is frequently underdiagnosed. Portable respiratory monitoring devices could facilitate early SDB screening in these patients. We estimated the prevalence of sleep apnea (SA) using a smartphone-based monitoring system in post-stroke patients and examined associations between respiratory indices, stroke severity and disability (NIHSS, mRS), and rehabilitation outcomes (motor and cognitive Functional Independence Measure; FIM). Consecutive patients admitted to inpatient rehabilitation within three months after a stroke underwent an overnight assessment with a smartphone-based respiratory monitoring device, which estimated the apnea-hypopnea index (AHI), mean and minimum SpO(2), time with SpO(2) < 94% and = 3% and >= 4%). Of the 104 screened patients, 59 were recruited, while 56 had valid recordings. Most patients (89%) had previously undiagnosed SA: 11% mild (AHI >= 5 and = 15 and = 30). Greater event burden and nocturnal hypoxemia were associated with older age, worse baseline disability (mRS), lower admission motor FIMs, and poorer rehabilitation metrics. Smartphone-based portable monitoring is an accessible, easy-to-use approach that may enable earlier identification of SA, particularly in individuals with substantial hypoxemia or respiratory event burden.

JTD Keywords: Apnea, Disturbance, Functional outcomes, Impact, Nocturnal hypoxemia, Portable device, Reliability, Risk, Sleep apnea, Sleep-disordered breathing, Stroke

Objective: Accurate detection of inspiratory onset and offset in the diaphragm electromyographic signal (EMGdi) is clinically relevant to assess patient-ventilator interaction in COPD patients undergoing non-invasive ventilation (NIV). Manual annotations are time-consuming and subject to inter-observer variability, highlighting the need for reliable automatic methods. Method: We developed a fully automatic algorithm to detect EMGdi activity cycles and their onset/offset timing in overnight NIV recordings. Four ECG suppression approaches were combined with root mean square (RMS) and fixed sample entropy (fSE) envelopes, and a novel bias correction strategy based on inspiratory-to-basal signal-to-noise ratio (I2BSNR) was introduced. Performance was compared with double-blind annotations from two independent experts. Results: In a cohort of 10 severe COPD patients (9212 annotated cycles), the best configuration (adaptive filtering with fSE exponential envelope) achieved F $1=0.96$ , with onset bias -28 ms (SD 270 ms) and offset bias + 120 ms (SD 292 ms). We show that fSE-based envelopes consistently outperform RMS in onset/offset detection, and that I2BSNR-based correction reduces systematic bias to within accepted clinical timing windows. Conclusions: The proposed method provides accurate and robust onset/offset detection of EMGdi during NIV in COPD patients. This enables reliable quantification of patient-ventilator asynchronies such as ineffective efforts and delayed cycling, offering direct clinical value for optimizing nightly ventilator settings in severe COPD. Clinical and Impact: Reliable detection of patient inspiratory activity offers a practical tool to guide real-time ventilator adjustments and reduce patient-ventilator asynchronies

JTD Keywords: Annotations, Asynchrony, Chronic obstructive pulmonary disease, Chronic obstructive pulmonary disease (copd), Electromyography, Emg, Filtering, Fixed sample entropy (fse)., Non-invasive ventilation (niv), Patient-ventilator asynchrony (pva), Recording, Reliability, Root mean square, Surface diaphragm electromyography (emgdi), Time, Timing, Ventilation, Ventilators

Astrocytes are central to brain homeostasis, supporting neuronal metabolism, synaptic activity, and the blood-brain barrier. With aging, these glial cells undergo molecular and functional changes that weaken support functions and promote neuroinflammation, contributing to neurodegeneration. Yet the systems-level mechanisms by which astrocytes respond to aging-related stressors remain poorly defined in human models. Because aging also heightens risk for cardiovascular disease, cognitive impairment, type 2 diabetes, and systemic inflammation, clarifying shared astrocytic pathways is critical for understanding brain-body crosstalk. Using an in vitro human astrocyte model exposed to sublethal oxidative stress (10 mu M H2O2) as a proxy for age-related cellular stress, we profiled transcriptomic changes and identified differentially expressed genes across antioxidant defenses, proteostasis, transcriptional regulation, vesicular trafficking, and inflammatory signaling. We then performed network-prioritization analyses on a curated human protein-protein interactome: one seeded with the astrocyte oxidative stress responsive genes and six with phenotype-associated gene sets (Alzheimer's disease, cardiovascular disease, cognitive impairment, type 2 diabetes, oxidative stress, and inflammation). Intersecting the top 5 % scoring genes from each run yielded a 127-gene core shared across all seven, enriched for proteostasis, DNA repair, mitochondrial regulation, and telomere and nuclear envelope maintenance. Structure-guided analyses highlighted vulnerable interfaces, including lamin A/C-lamin B1, alpha-actinin-filamins, 14-3-3 dimers, and aminoacyl-tRNA synthetase assemblies, where pathogenic variants are predicted to destabilize or aberrantly stabilize protein interactions. Structure-based interface predictions also highlight potential interactions between amyloid precursor protein (APP) and valosin-containing protein (VCP), and between p53 and 14-3-3 zeta, poten-tially linking proteostasis and stress signaling. Together, these analyses identify a conserved astrocyte-centered network signature that may relate neurodegenerative and cardiovascular processes, and prioritize structurally testable candidates for biomarker and intervention hypothesis testing.

JTD Keywords: 14-3-3-zeta, Aging-associated proteomic remodeling, Astrocytic vulnerability networks, Crosstalk, Disease, Dysfunction, Insights, Interactome, Interfaces, Mutations, Network-based gene-disease prioritization, Neurodegeneration-cardiovascular disease, Oxidative stress-responsive astrocyte pathways, Phosphorylation, Prediction, Proteostasis and mitochondrial dysfunction, Receptor, Structurally vulnerable proteinprotein, Structure-guided variant impact prediction, Telomere and nuclear envelope integrity, Update

Gold nanoparticles (GNP) are highly valuable in nanotechnology due to their biocompatibility and unique physicochemical properties, which make them attractive as nanocarriers for targeted drug delivery. In the context of neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD), GNP hold promise for reducing the toxicity of Amyloid-beta peptide (A beta) aggregates. However, a major challenge in developing new therapies for NDDs lies in the limited reliance on animal models and the difficulty of crossing the blood-brain barrier (BBB). This study investigates the effects of GNP on A beta toxicity using a human-based BBB-organ-on-a-chip model (BBBoC), mimicking the 3D cellular architecture of the BBB under both normal and pathological conditions. We rationally designed a novel nanosystem functionalized with the peptide D3, which functions both as a selective A beta toxicity inhibitor and a BBB-targeting agent. The results show that GNP can cross the BBB, reduce the A beta- induced cytotoxicity, and promote the maintenance of the BBB integrity. Moreover, controlling the shape of GNP further enhanced their protective effect. Overall, this work highlights the feasibility of rationally designed GNP as a promising therapeutic strategy for AD, evaluated through a more reliable and predictive human-relevant model.

JTD Keywords: Alzheimer's disease, Amyloid-beta peptide, Arginine-rich, Blood-brain barrier, Elegans, Endothelial-cells, Gold nanoparticles, Hypothesis, Impact, Integrity, Microfluidic, Nanorods, Oligomers, Organ-on-a-chip, Permeability, Toxicity

In this study, a nasal spray was formulated and tested co-loading grape seed extract, thymol, and camphor in penetration enhancer containing vesicles (PEVs) tailored to synergistically protect the nasal mucosa against oxidative stress and bacterial colonization. Based on their previously demonstrated effects, PEVs were prepared with propylene glycol (PG) and further enriched with carrageenan to promote muco-adhesion. The mean diameter of PG-PEVs was similar to 177 nm, and that of carrageenan PG-PEVs was similar to 194 nm. The polydispersity index ranged from 0.25 to 0.27, confirming the homogeneity of the dispersions. The zeta potential was significantly negative (similar to- 63 mV) and the entrapment efficiency was similar to 88 %, irrespective of vesicle composition. Sprayability studies disclosed that both PG-PEVs and carrageenan-PG-PEVs generated droplets larger than 10 mu m, thus appropriate for the deposition in the nasal cavity. Regional nasal deposition studies, carried out with a realistic nasal replica, highlighted that formulation droplets were deposited in the vestibule and turbinate areas of the nose. The ability of formulations to inhibit protein denaturation confirmed their anti-inflammatory effects. In vitro study with A549 and CuFi-1 cells, underlined that PG-PEVs and especially carrageenan PG-PEVs were nontoxic (viability similar to 140 %) and effectively counteracted cell apoptosis caused by hydrogen peroxide, restoring healthy conditions. The in vivo study in mice demonstrated that grape seed extract, thymol, and camphor-loaded carrageenan PG-PEVs were highly effective in counteracting the proliferation of Staphylococcus aureus.

JTD Keywords: Ability, Chitosan, Chronic rhinosinusitis, Droplet distribution, Dru, Grape seeds, In-vitro, Liposomes, Nasal spray, Oxidative stress, Pg-pevs and carrageenan-pg-pevs, Phospholipid vesicles, Polyphenols, Staphylococcus aureu, Systems, Therapy

The class Ia ribonucleotide reductase (RNR), encoded by the nrdAB operon in Pseudomonas aeruginosa, has a long 5' untranslated region (5' UTR) whose regulatory role remains poorly understood. In this study, we investigated the functional significance of the nrdAB 5' UTR using a comprehensive set of bioinformatic and experimental approaches, including gene expression profiling, relative protein levels, and in vivo infection assays in the Galleria mellonella animal model. Our results demonstrate that the 5' UTR negatively regulates nrdA expression by reducing transcript levels, decreasing mRNA stability and limiting protein abundance. Truncation of this region led to increased expression, particularly during the stationary phase, suggesting that this region may contribute to modulating RNR activity during the stationary phase, when dNTP demand is reduced. In vivo, the Delta 5' UTR mutant exhibited reduced virulence compared to the wild-type strain, accompanied by elevated nrdA mRNA levels and a modest decrease in nrdJ expression. Although these changes may contribute to altered RNR regulation during infection, they are unlikely to fully explain the observed phenotype, indicating that additional factors may be involved. Although no specific regulatory elements within or acting upon the 5' UTR were probed under the tested conditions, the possibility of such mechanisms cannot be excluded, and further studies will be needed to elucidate the underlying molecular mechanisms. Overall, our findings demonstrate that the 5' UTR plays a regulatory role in modulating nrdAB operon expression and contributes to maintaining RNR system homeostasis in P. aeruginosa.

JTD Keywords: 5' utr, galleria mellonella, Bacteria, Expression, Identification, Metabolism, Mrna stability, Prediction, Proteins, Relative protein levels, Ribonucleotide reductase, Sequence, Tool, Transcript levels, Transcription

Early prediction of weaning outcomes in mechanically ventilated patients has significant potential to influence the duration of treatment as well as associated morbidity and mortality. This study aimed to investigate the utility of signal analysis using electromyographic diaphragm (EMG) and electrocardiography (ECG) signals to classify the success or failure of weaning in mechanically ventilated patients. Electromyographic signals of 40 subjects were recorded using 5-channel surface electrodes placed around the diaphragm muscle, along with an ECG recording through a 3-lead Holter system during extubation. EMG and ECG signals were recorded from mechanically ventilated patients undergoing weaning trials. Linear and nonlinear signal analysis techniques were used to assess the interaction between diaphragm muscle activity and cardiac activity. Supervised machine learning algorithms were then used to classify the weaning outcomes. The study revealed clear differences in diaphragmatic and cardiac patterns between patients who succeeded and failed in the weaning trials. Successful weaning was characterised by a higher ECG-derived respiration amplitude, whereas failed weaning was characterised by an elevated EMG amplitude. Furthermore, successful weaning exhibited greater oscillations in diaphragmatic muscle activity. Spectral analysis and parameter extraction identified 320 parameters, of which 43 were significant predictors of weaning outcomes. Using seven of these parameters, the Naive Bayes classifier demonstrated high accuracy in classifying weaning outcomes. Surface electromyographic and electrocardiographic signal analyses can predict weaning outcomes in mechanically ventilated patients. This approach could facilitate the early identification of patients at risk of weaning failure, allowing for improved clinical management.

JTD Keywords: Cardiorespiratory characterisation, Classification, Coherence, Diaphragm, Ecg, Electromyography, Emg, Heart-rate-variability, Mechanical ventilation, Naive bayes classifier, Predictor, Signal analysis, Surface electromyography, Time-series analysis, Time-varying spectral analysis, Weaning outcome

Cationic coatings on titanium surfaces are a promising approach for dental and biomedical implants due to their low-cost antimicrobial effect and no need for antibiotics. These coatings are applied on hydroxylated (-OH) surfaces using silanes, such as 3-aminopropyltriethoxysilane (APTES). However, it is unclear whether the concentration of this organofunctional compound affects surface charge or potential toxicity. This study investigated how different concentrations of APTES in cationic coatings on titanium samples influence electrostatic behavior and interactions with bacteria and mesenchymal stem cells (MSCs). Titanium discs served as controls (Ti group) and were first treated by plasma electrolytic oxidation (PEO) to generate -OH groups (PEO group). Subsequently, APTES was applied at 83.8, 167.6, and 251.4 mM, forming PEO+APTES0.3, PEO+APTES0.6, and PEO+APTES0.9 groups, respectively. Surfaces were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), contact angle, Zeta potential, and profilometry. Microbiological assays assessed initial bacterial adhesion (1 h) and biofilm formation (24 h) using Staphylococcus aureus and Escherichia coli. Cell metabolism was assessed on days 1, 3, and 8, while cell viability was assessed on days 1 and 3 using mesenchymal stem cells. PEO-treated surfaces showed porous morphology, and silanization increased roughness and shifted surfaces toward hydrophobicity. Amines and surface charge changes were confirmed by XPS and Zeta potential. Increasing APTES concentration did not proportionally increase cation number. Crystalline hydroxyapatite oxides were identified following the electrochemical process. SEM, EDS, and FTIR confirmed treatment stability after 28 days of immersion, while tribological tests indicated improved performance for PEO-treated groups. Cationic coatings reduced bacterial adhesion by up to 65%, decreased biofilm Log10 values, and increased dead bacteria proportion. Biocompatibility was confirmed by metabolism and cell viability tests, with the group with lower APTES concentration showing the best performance on day 8, with an 80% higher cell metabolism than day 1. On the other hand, higher concentrations of APTES resulted in reduced cell metabolism. These findings indicate, for the first time, that APTES concentration does not affect electrostatic properties but that lower concentrations are required for cytocompatible cationic coatings.

JTD Keywords: Adhesion, Biofilms, Cationic coating, Cell proliferation, Coatings, Electrostatic interactions, Implant surfaces, In-vitro, Nanoparticles, Osteoblasts, Oxidation, Self-assembled monolayers, Thin-films, Titanium, Wettability

Nucleic acid hybridization in bimolecular and folding reactions is a fundamental kinetic process susceptible to water solvation, counterions, and chemical modifications with intricate enthalpy-entropy compensation effects. Such effects hinder the typically weak temperature dependencies of enthalpies and entropies quantified by the heat capacity change upon duplex formation. Using a temperature-jump optical trap, we investigate the folding thermodynamics and kinetics of DNA hairpins of varying stem sequences and loop sizes in the temperature range of 5-40 circle C. From a kinetic analysis and using a Clausius-Clapeyron equation in force, we derive the hybridization heat capacity changes Delta Cp per GC and AT bp, finding 36 +/- 3 and 29 +/- 3 cal/(mol K), respectively. The almost equal values imply similar degrees of freedom arrest upon GC and AT bp formation during duplex formation. Folding kinetics on DNA hairpins of varying loop sizes show that the transition states (TS) in duplex formation have high free energies but low Delta Cp values relative to the native state. Consequently, TS have low configurational entropy in agreement with the funnel-like energy landscape hypotheses. Our study underlines the validity of general principles in the hybridization and folding of nucleic acids determined by the TS's Delta Cp values.

JTD Keywords: Binding, Duplex, Force, Free-energy-landscape, Heat-capacity changes, Kinetics, Protein, Stability, Thermodynamic, Transition-state

Antifouling coatings are vital to enhance the performance of medical devices, aiming to mitigate bodily reactions by shielding their surface. Despite significant advancements in antifouling coatings, like those based on zwitterionic monomers and hydroxyl-functionalized (meth)acrylamides, limitations like decreased antifouling properties after functionalization and complement system activation hinder their application in blood. Here, a novel class of ultrathin surface-attached hydrogels is presented, consisting of hydrophilic non-charged green solvent-based monomers and preventing protein adsorption while offering on-demand degradability. Unlike the best antifouling brushes, the coatings are easily applicable, unaffected by charges, and free of complement system-activating groups. The hydrogels are formed using copolymers of N,N-dimethyl lactamide acrylate (DMLA) and benzophenone acrylate (BPA). Moreover, 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) is incorporated to introduce hydrolyzable ester. The coating of state-of-the-art devices is demonstrated with X-ray photoelectron spectroscopy (XPS), analyze surface energy components, and confirm their antifouling properties with surface plasmon resonance (SPR). The coatings are non-cytotoxic toward MRC-5 fibroblasts, exhibit repellency against methicillin-resistant Staphylococcus aureus (MRSA), and effectively prevent thrombus formation on devices in blood. This work establishes a versatile platform for next-generation coatings in medical and industrial applications, matching the antifouling efficiency of the most advanced solutions and offering regeneration of substrates by erasing the coating.

JTD Keywords: Adhesion, Antifouling, Biocompatibility, Blood-plasma, Coating, Coatings, Contact, Copolymers, Degradability, Energy components, Green solvent, Hemocompatibilit, Hydrogel, Polyethylene oxide, Polymer brushes, Protein adsorption, Thi

Cancer ranks as a leading cause of death worldwide, with liver cancer being one of the most commonly diagnosed. Currently, several molecules are being studied in order to find new therapeutic options that can reduce cancer recurrence rate and increase patient survival. This study proposes PEGylated liposomes for the delivery of a newly synthesized aminohydroxy sulfonamide, BupM-NH2, which has shown dose-dependent cytotoxicity towards hepatic tumor cells. The prepared PEG-liposomes were nanosized, spherical, and unilamellar, as shown by light scattering data and cryo-TEM micrographs. The physical stability of the PEG-liposomes was preserved when tested in simulated body fluids. Similar results were found for the storage stability evaluation. Furthermore, the PEG-liposomes efficiently entrapped BupM-NH2 and modulated its release. The antitumor activity of BupM-NH2 in PEG-liposomes was assessed in vitro in hepatocarcinoma cells. The viability assay showed that PEG-liposomes were able to control the release of BupM-NH2 over time and induce the death of HepG2 cells at a concentration about two-times lower than that required by free BupM-NH2 (IC50: 33.31 vs. 57.05 mu M). Furthermore, the liposomal formulation showed a less cytotoxic effect against non-cancerous cell line (IHH) compared to the free molecule (IC50 > 200 vs. 106.9 mu M), encouraging further investigation to confirm its effective and safe use.

JTD Keywords: Aminohydroxy sulfonamide, Anticancer formulation, Cancer, Delivery-systems, Drug-delivery, Hepatic cancer cells, Parenteral delivery, Peg-modified liposomes, Pegylated liposomes, Stability

Heterogeneity and the absence of a tumor microenvironment (TME) in traditional patient-derived organoid (PDO) cultures limit their effectiveness for clinical use. Here, Embedded Bioprinting-enabled Arrayed PDOs (Eba-PDOs) featuring uniformly arrayed colorectal cancer (CRC) PDOs within a recreated TME is presented. This model faithfully reproduces critical TME attributes, including elevated matrix stiffness (approximate to 7.5 kPa) and hypoxic conditions found in CRC. Transcriptomic and immunofluorescence microscopy analysis reveal that Eba-PDOs more accurately represent actual tissues compared to traditional PDOs. Furthermore, Eba-PDO effectively capture the variability of CEACAM5 expression-a critical CRC marker-across different patients, correlating with patient classification and differential responses to 5-fluorouracil treatment. This method achieves an uniform size and shape within PDOs from the same patient while preserving distinct morphological features among those from different individuals. These features of Eba-PDO enable the efficient development of a label-free, morphology-based predictive model using supervised learning, enhancing its suitability for clinical applications. Thus, this approach to PDO bioprinting is a promising tool for generating personalized tumor models and advancing precision medicine.

JTD Keywords: Association, Cancer, Carcinoembryonic antigen-expression, Colorectal cancer, Cultur, Differential expression, Embedded bioprinting, Extracellular-matrix, Inter-patient variability, Patient-derived tumor organoid, Stem-cell, Stiffness, Supervised learning, Tissues, Tumor matrix stiffnes

Cell membranes play a key role in bottom-up synthetic biology, as they enable interaction control, transport, and other essential functions. These ultra-thin, flexible, yet stable structures form through the self-assembly of lipids and proteins. While liposomes are common mimics, their synthetic membranes often fail to replicate natural properties due to poor structural control. To address this, pepticombs are introduced, a new family of supramolecular building blocks. They are synthesized by regularly appending anionic surfactants with lipid-long alkyl tails to cationic amino acid residues of recombinant elastin-like supercharged unfolded polypeptides (SUPs). Using microscopy techniques and molecular dynamics simulations, the formation of giant unilamellar vesicles, termed pepticombisomes, is demonstrated and their membrane properties are characterized. The molecular topology of pepticombs allows for precise mimicry of membrane thickness and flexibility, beyond classic polymersomes. Unlike the previously introduced ionically-linked comb polymers, all pepticombs exhibit a uniform degree of polymerization, composition, sequence, and spontaneous curvature. This uniformity ensures consistent hydrophobic tail distribution, facilitating intermolecular hydrogen bonding within the backbone. This generates elastic heterogeneities and the concomitant formation of non-icosahedral faceted vesicles, as previously predicted. Additionally, pepticombisomes can incorporate functional lipids, enhancing design flexibility.

JTD Keywords: Biomimetic synthesis, Bottom-up synthetic biology, Cholesterol, Dynamics, Glycodendrimersomes, Janus dendrimers, Lipids, Nanoscal, Organization, Polymersome membranes, Protein, Stability, Supercharged peptides, Synthetic cells, Vesicle

Acute respiratory distress syndrome (ARDS) is a severe pulmonary condition that often requires mechanical ventilation (MV) to ensure adequate gas exchange and minimize ventilator-induced lung injury. This study compares statistical descriptors derived from respiratory time series with nonlinear variability metrics obtained from Poincar & eacute; plots to predict the outcomes of the weaning process in mechanically ventilated patients. The database, which includes respiratory flow recordings from 243 patients who underwent a standardized 30-minute spontaneous breathing test (SBT), was used to validate the classification models. Patients were categorized into three clinical outcome groups: successful weaning (n = 132), failed weaning (n = 88), and reintubation within 48 hours after completion of the trial (n = 23). Features were selected using nonparametric statistical tests and correlation analysis, eliminating redundancy and retaining discriminative variables. Two machine learning (ML) classifiers, random forest and feedforward neural network, were designed to identify patients belonging to each of the three clinical outcome groups. Model performance was assessed using stratified hold-out cross-validation repeated over 150 iterations, with hyperparameters optimized using Bayesian methods. The random forest classifier using Poincar & eacute; descriptors achieved a mean accuracy of 90.4% and higher F1-scores across all groups, including those who required reintubation. These findings suggest that Poincar & eacute;-based variability metrics, in combination with ensemble learning, may enhance the accurate prediction of MV weaning outcomes in ARDS patients.

JTD Keywords: Heart-rate-variability, Mechanical ventilation,weaning,acute respiratory distress syndrome,poincar & eacute, Plot, Plot,mechanical ventilation,weaning,acute respiratory distress syndrome,poincar & eacute, Successful extubation

The mechanics of the extracellular matrix (ECM) determine cell activity and fate through mechanoresponsive proteins including Yes-associated protein 1 (YAP). Rigidity and viscous relaxation have emerged as the main mechanical properties of the ECM steering cell behavior. However, how cells integrate coexisting ECM rigidity and viscosity cues remains poorly understood, particularly in the high-stiffness regime. Here, we have exploited engineered stiff viscoelastic protein hydrogels to show that, contrary to current models of cell-ECM interaction, substrate viscous energy dissipation attenuates mechanosensing even when cells are exposed to higher effective rigidity. This unexpected behavior is however readily captured by a pull-and-hold model of molecular clutch-based cell mechanosensing, which also recapitulates opposite cellular response at low rigidities. Consistent with predictions of the pull-and-hold model, we find that myosin inhibition can boost mechanosensing on cells cultured on dissipative matrices. Together, our work provides general mechanistic understanding on how cells respond to the viscoelastic properties of the ECM.

JTD Keywords: Adhesion, Animals, Defines, Dynamics, Elasticity, Extracellular matrix, Force, Humans, Hydrogels, Mechanotransduction, Mechanotransduction, cellular, Stability, Tali, Viscosity, Yap/taz

Permanently polarized biphasic calcium phosphate composed of hydroxyapatite and brushite (pp-HAp/Bru), which is prepared by applying the thermally stimulated polarization treatment to calcined HAp, is used as a sustainable catalyst to transform CO2 into value-added products. In this work, the stability of pp-HAp/Bru is studied from structural, electrical, and catalytic perspectives, applying a thermal depolarization process with temperatures (T-d) ranging from 200 to 1000 degrees C. Results show that the Bru phase is not stable when T-d = 600 degrees C. Besides, the electrical resistance and capacitance of the pp-HAp/Bru increase with T-d, evidencing the progressive electrical depolarization of the material. Thermal depolarization also influences the specific orientation of the OH- ions, which is partially lost (approximate to 50%). All such changes affect the catalytic efficiency of pp-HAp/Bru, which is proven using a reaction that transforms CO2 gas into acetic acid and formic acid. Results show that the total reaction yield linearly decreases with increasing T-d. Based on such observations, a simple process is designed that allows the reconstitution of the structure and restores the activity of such green catalysts.

JTD Keywords: Co-2 fixation, Electrical depolarization, Green catalysts, Interface stability, Thermal depolarizatio

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.© 2024 The Authors. Small Methods published by Wiley-VCH GmbH.

JTD Keywords: 3d cancer cell uptake, Cancer cells, Cell culture, Cell uptake, Cellular uptake, Diseases, Ecm penetration, Extracellular matrices, Extracellular matrix penetration, Functional polymers, Hydrogen bonds, Medical applications, Microfluidics, Microstructure, Nanoparticles, Polymeric nanoparticles, Scpns, Single chains, Single-chain polymeric nanoparticle, Stability, Tumor-on-a-chip, Tumors

The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Models for the study of the physiological processes affecting the BBB, as well as predicting the permeability of therapeutic substances for neurological and neurovascular pathologies, are highly desirable. Existing models, such as Transwell utilizing-models, do not mimic the extracellular environment of the BBB with their stiff, semipermeable, non-biodegradable membranes. To help overcome this, we engineered electrospun membranes from poly L-lactic acid in combination with a nanometric coating of poly(ethyl acrylate) (PEA) that drives fibrillogenesis of fibronectin, facilitating the synergistic presentation of both growth factors and integrin binding sites. Compared to commercial semi-porous membranes, these membranes significantly improve the expression of BBB-related proteins in brain endothelial cells. PEA-coated membranes in combination with different growth factors and extracellular protein coatings reveal nerve growth factor (NGF) and fibroblast growth factor (FGF-2) caused formation of better barriers in vitro. This BBB model offers a robust platform for studying key biochemical factors influencing barrier formation that marries the simplicity of the Transwell model with the highly tunable electrospun PEA-fibronectin membranes. This enables the generation of high-throughput drug permeability models without the need of complicated co-culture conditions. The blood-brain barrier (BBB) tightly regulates substance transport between the bloodstream and the brain. Here a simple model of the BBB that allows culture of endothelial cells on growth-factor functionalised membranes is introduced. This novel in vitro model of the BBB offers a robust platform for studying key barriergenic biochemical factors influencing barrier formation without the use of the complicated co-culture conditions. image

JTD Keywords: Animals, Bbb, Blood-brain barrier, Densit, Differentiation, Ecm, Electrospinning, Endothelial cells, Endothelial-cell lines, Expression, Fiber diameter, Fibroblast-growth-factor, Growth factors, Humans, In vitro mode, In vitro model, Membranes, artificial, Models, biological, Morphology, Permeability, Poly(l-lactic acid), Poly(lactide), Polyesters, Proteins

Direct ink writing (DIW) is a promising technology for the fabrication of personalized bone grafts, as it enables the customization of their geometrical conformation with high reproducibility and is compatible with the use of self-setting calcium-deficient hydroxyapatite inks. However, the scaffolds obtained by DIW consist mostly of convex filaments, which is a limitation since concave surfaces are known to promote bone regeneration in vivo. In this work, we explore the use of triply periodic minimal surface (TPMS) designs in DIW of calcium phosphate self-hardening inks as a strategy to obtain scaffolds with controlled concave macropores. The limitations of the printing parameters with high ceramic-loaded inks using DIW resulted in only 20% nominal porosity for gyroid-, diamond-, and Schwarz-based structures. The inherent layered pores from TPMS geometries enabled concavities typically unattainable via DIW, bearing substantial implications for subsequent osteoinductive capabilities. Although the mechanical properties were lower in the TPMS-based scaffolds than in the orthogonal patterned ones, the blood permeability of TPMS-based structures was higher. The concave pore architecture enhanced the osteogenic potential of the and mineralization.

JTD Keywords: Alkaline-phosphatase, Biomimetic hydroxyapatite, Biomorphic structures, Blood analog fluid, Bone, Bone scaffold, Concavity, Defects, Direct ink writing, Mineralization, Minimal-surfaces, Permeability, Pore architectur, Shap, Stress, Tissue

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth. The mechanical properties of heterogeneous cell populations in colorectal tumors and the relevance to cancer metastasis remain not fully understood. Here, the authors suggest that the variations in malignant phenotypes between LGR5-positive cancer stem cells and LGR5-negative cells could be due to their distinct mechanical phenotypes observed in vitro, determined by the membrane to cortex attachment proteins Ezrin/Radixin/Moesin.

JTD Keywords: , , Adhesion, Deformability, E-cadherin, Erm proteins, Expression, Metastasis, Organization, Plasticit, Stem-cells, Tumor-cells

Exposure of experimental rodents to controlled cycles of light, food, and temperature is important when investigating alterations in circadian cycles that profoundly influence health and disease. However, applying such stimuli simultaneously is difficult in practice. We aimed to design, build, test, and open-source describe a simple device that subjects a conventional mouse cage to independent cycles of physiologically relevant environmental variables. The device is based on a box enclosing the rodent cage to modify the light, feeding, and temperature environments. The device provides temperature-controlled air conditioning (heating or cooling) by a Peltier module and includes programmable feeding and illumination. All functions are set by a user-friendly front panel for independent cycle programming. Bench testing with a model simulating the CO2 production of mice in the cage showed: a) suitable air renewal (by measuring actual ambient CO2), b) controlled realistic illumination at the mouse enclosure (measured by a photometer), c) stable temperature control, and d) correct cycling of light, feeding, and temperature. The cost of all the supplies (retail purchased by e-commerce) was <300 US$. Detailed technical information is open-source provided, allowing for any user to reliably reproduce or modify the device. This approach can considerably facilitate circadian research since using one of the described low-cost devices for any mouse group with a given light-food-temperature paradigm allows for all the experiments to be performed simultaneously, thereby requiring no changes in the light/temperature of a general-use laboratory. 1 Introduction

JTD Keywords: Animal experiment, Animal model, Animal research, Article, Circadian alteration, Circadian rhythm, Commercial phenomena, Controlled study, Cycling, Energy consumption, Energy-expenditure, Experimental model, Feeding, Food, Food availability, Illumination, Intermittent fasting, Light, Light cycle, Light dark cycle, Mouse, Nonhuman, Open source technology, Open-source hardware, Performance, Photography, Research, Rhythms, Rodent, Temperature, Temperature cycle

The use of nanocarriers in medicine, so-called nanomedicine, is one of the most innovative strategies for targeting drugs at the action site and increasing their activity index and effectiveness. Phytomedicine is the oldest traditional method used to treat human diseases and solve health problems. The recent literature on the treatment of malaria infections using nanodelivery systems and phytodrugs or supplements has been analyzed. For the first time, in the present review, a careful look at the considerable potential of nanomedicine in promoting phytotherapeutic efficacy was done, and its key role in addressing a translation through a significant reduction of the current burden of malaria in many parts of the world has been underlined.

JTD Keywords: antiplasmodial activity, bioavailability, chloroquine, combination therapy, discovery, drug-delivery, drug-delivery systems, nanocapsules, nanomedicine, natural molecules, pharmacokinetics, phytomedicine, plasmodium-falciparum, Artemisinin-based combination therapy, Drug-delivery systems, Nanomedicine, Natural molecules, Phytomedicine, Solid lipid nanoparticles

General porins are nature's sieving machinery in the outer membrane of Gram-negative bacteria. Their unique hourglass-shaped architecture is highly conserved among different bacterial membrane proteins and other biological channels. These biological nanopores have been designed to protect the interior of the bacterial cell from leakage of toxic compounds while selectively allowing the entry of the molecules needed for cell growth and function. The mechanism of transport through porins is of utmost and direct interest for drug discovery, extending toward nanotechnology applications for blue energy, separations, and sequencing. Here we present a theoretical framework for analysing the filter of general porins in relation to translocating molecules with the aid of enhanced molecular simulations quantitatively. Using different electrostatic probes in the form of a series of related molecules, we describe the nature of this filter and how to finely tune permeability by exploiting electrostatic interactions between the pore and the translocating molecule. Eventually, we show how enhanced simulations constitute today a valid tool for characterising the mechanism and quantifying energetically the transport of molecules through nanopores. General porins are nature's sieving machinery in the outer membrane of Gram-negative bacteria. In the diffusive transport process of molecules, electrostatic interactions can help to decrease the entropic free energy barrier.

JTD Keywords: Channel, Diffusion barrier, Electric-field, Molecular-dynamics, Outer-membrane permeability, Permeation, Porins, Simulations, Translocation, Transport

Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.

JTD Keywords: biocompatibility, cytotoxicity, delivery, dental implants, prevention, release, stability, surface coating, titanium, zirconia, Antimicrobial peptide, Biocompatibility, Dental implants, Peri-implantitis, Surface coating, Titanium

Alzheimer's disease is characterized by a combination of several neuropathological hallmarks, such as extracellular aggregates of beta amyloid (Aβ). Numerous alternatives have been studied for inhibiting Aβ aggregation but, at this time, there are no effective treatments available. Here, we developed the tri-component nanohybrid system AuNPs@POM@PEG based on gold nanoparticles (AuNPs) covered with polyoxometalates (POMs) and polyethylene glycol (PEG). In this work, AuNPs@POM@PEG demonstrated the inhibition of the formation of amyloid fibrils, showing a 75% decrease in Aβ aggregation in vitro. As it is a potential candidate for the treatment of Alzheimer's disease, we evaluated the cytotoxicity of AuNPs@POM@PEG and its ability to cross the blood-brain barrier (BBB). We achieved a stable nanosystem that is non-cytotoxic below 2.5 nM to human neurovascular cells. The brain permeability of AuNPs@POM@PEG was analyzed in an in vitro microphysiological model of the BBB (BBB-on-a-chip), containing 3D human neurovascular cell co-cultures and microfluidics. The results show that AuNPs@POM@PEG was able to cross the brain endothelial barrier in the chip and demonstrated that POM does not affect the barrier integrity, giving the green light to further studies into this system as a nanotherapeutic.

JTD Keywords: beta-amyloid, blood-brain barrier organ-on-a-chip, cellular uptake, citrate, cytotoxicity, electrocatalytic reduction, gold nanoparticles, hypothesis, nanorods, polyoxometalates, size, stability, surface, Alzheimers-disease, Blood–brain barrier organ-on-a-chip, Gold nanoparticles, Nanovehicle, Polyoxometalates, Β-amyloid

The small intestine is a complex organ with a characteristic architecture and a major site for drug and nutrient absorption. The three-dimensional (3D) topography organized in finger-like protrusions called villi increases surface area remarkably, granting a more efficient absorption process. The intestinal mucosa, where this process occurs, is a multilayered and multicell-type tissue barrier. In vitro intestinal models are routinely used to study different physiological and pathological processes in the gut, including compound absorption. Still, standard models are typically two-dimensional (2D) and represent only the epithelial barrier, lacking the cues offered by the 3D architecture and the stromal components present in vivo, often leading to inaccurate results. In this work, we studied the impact of the 3D architecture of the gut on drug transport using a bioprinted 3D model of the intestinal mucosa containing both the epithelial and the stromal compartments. Human intestinal fibroblasts were embedded in a previously optimized hydrogel bioink, and enterocytes and goblet cells were seeded on top to mimic the intestinal mucosa. The embedded fibroblasts thrived inside the hydrogel, remodeling the surrounding extracellular matrix. The epithelial cells fully covered the hydrogel scaffolds and formed a uniform cell layer with barrier properties close to in vivo. In particular, the villus-like model revealed overall increased permeability compared to a flat counterpart composed by the same hydrogel and cells. In addition, the efflux activity of the P-glycoprotein (P-gp) transporter was significantly reduced in the villus-like scaffold compared to a flat model, and the genetic expression of other drugs transporters was, in general, more relevant in the villus-like model. Globally, this study corroborates that the presence of the 3D architecture promotes a more physiological differentiation of the epithelial barrier, providing more accurate data on drug absorbance measurements.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: 3d architecture, alkaline-phosphatase, caco-2 cells, culture, drug development, efflux proteins, gene-expression, human-colon, intestinal absorption, intestinal models, microenvironment, paracellular transport, permeability, photopolymerization, villi, 3d architecture, 3d bioprinting, Drug development, In-vitro, Intestinal absorption, Intestinal models, Photopolymerization, Villi

In order to exploit the pharmacological potential of natural bioactive molecules with low water solubility, such as curcumin, it is necessary to develop formulations, such as amorphous polymer dispersions, which allow a constant release rate and at the same time avoid possible toxicity effects of the crystalline form of the molecule under scrutiny. In this study, polymer dispersions of curcumin were obtained in PADAS, a biodegradable semicrystalline copolymer based on 1,12-dodecanediol, sebacic acid and alanine. The dispersions were fully characterized by means of differential scanning calorimetry and broadband dielectric spectroscopy, and the drug release profile was measured in a simulated body fluid. Amorphous homogeneous binary dispersions were obtained for curcumin mass fraction between 30 and 50%. Curcumin has significantly higher glass transition temperature Tg (≈ 347 K) than the polymer matrix (≈274-277 K depending on the molecular weight), and dispersions displayed Tg's intermediate between those of the pure amorphous components, implying that curcumin acts as an effective antiplasticizer for PADAS. Dielectric spectroscopy was employed to assess the relaxation dynamics of the binary dispersion with 30 wt% curcumin, as well as that of each (amorphous) component separately. The binary dispersion was characterized by a single structural relaxation, a single Johari-Goldstein process, and two local intramolecular processes, one for each component. Interestingly, the latter processes scaled with the Tg of the sample, indicating that they are viscosity-sensitive. In addition, both the pristine polymer and the dispersion exhibited an interfacial Maxwell-Wagner relaxation, likely due to spatial heterogeneities associated with phase disproportionation in this polymer. The release of curcumin from the dispersion in a simulated body fluid followed a Fickian diffusion profile, and 51% of the initial curcumin content was released in 48 h.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: antioxidant, bioavailability, dielectric spectroscopy, domain havriliak-negami, glass transition temperature, kinetic stability, molecular mobility, nm pores, phase-behavior, physical stability, release kinetics, temperature, thermodynamic quantities, time, Amorphous formulations, Dielectric spectroscopy, Glass transition temperature, Kinetic stability, Kohlrausch-williams-watts, Molecular mobility, Release kinetics

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

A large portion of the elderly population are affected by cardiovascular diseases. Early prognosis of cardiomyopathies remains a challenge. The aim of this study was to classify cardiomyopathy patients by their etiology based on significant indexes extracted from the characterization of the baroreflex mechanism in function of the influence of the cardio-respiratory activity over the blood pressure. Forty-one cardiomyopathy patients (CMP) classified as ischemic (ICM-24 patients) and dilated (DCM-17 patients) were considered. In addition, thirty-nine control (CON) subjects were used as reference. The beat-to-beat (BBI) time series, from the electrocardiographic (ECG) signal, the systolic (SBP), and diastolic (DBP) time series, from the blood pressure signal (BP), and the respiratory time (TT), from the respiratory flow (RF) signal, were extracted. The three-dimensional representation of the cardiorespiratory and vascular activities was characterized geometrically, by fitting a polygon that contains 95% of data, and by statistical descriptive indices. DCM patients presented specific patterns in the respiratory response to decreasing blood pressure activity. ICM patients presented more stable cardiorespiratory activity in comparison with DCM patients. In general, CMP shown limited ability to regulate changes in blood pressure. In addition, patients also shown a limited ability of their cardiac and respiratory systems response to regulate incremental changes of the vascular variability and a lower heart rate variability. The best classifiers were used to build support vector machine models. The optimal model to classify ICM versus DCM patients achieved 92.7% accuracy, 94.1% sensitivity, and 91.7% specificity. When comparing CMP patients and CON subjects, the best model achieved 86.2% accuracy, 82.9% sensitivity, and 89.7% specificity. When comparing ICM patients and CON subjects, the best model achieved 88.9% accuracy, 87.5% sensitivity, and 89.7% specificity. When comparing DCM patients and CON subjects, the best model achieved 87.5% accuracy, 76.5% sensitivity, and 92.3% specificity. In conclusion, this study introduced a new method for the classification of patients by their etiology based on new indices from the analysis of the baroreflex mechanism.Copyright © 2023 Rodriguez, Schulz, Voss, Herrera, Benito and Giraldo.

JTD Keywords: abnormalities, blood pressure variability, cardio-respiratory variability, dilated cardiomyopathy, disease, heart-failure secondary, ischemic cardiomyopathy, ischemic-dilated cardiomyopathy, morphology-relative change, Baroreflex activity, Blood pressure variability, Cardio-respiratory variability, Cheyne-stokes respiration, Ischemic-dilated cardiomyopathy, Morphology-relative change

The low endogenous regenerative capacity of the heart,added tothe prevalence of cardiovascular diseases, triggered the advent ofcardiac tissue engineering in the last decades. The myocardial nicheplays a critical role in directing the function and fate of cardiomyocytes;therefore, engineering a biomimetic scaffold holds excellent promise.We produced an electroconductive cardiac patch of bacterial nanocellulose(BC) with polypyrrole nanoparticles (Ppy NPs) to mimic the naturalmyocardial microenvironment. BC offers a 3D interconnected fiber structurewith high flexibility, which is ideal for hosting Ppy nanoparticles.BC-Ppy composites were produced by decorating the network of BC fibers(65 & PLUSMN; 12 nm) with conductive Ppy nanoparticles (83 & PLUSMN; 8 nm).Ppy NPs effectively augment the conductivity, surface roughness, andthickness of BC composites despite reducing scaffolds' transparency.BC-Ppy composites were flexible (up to 10 mM Ppy), maintained theirintricate 3D extracellular matrix-like mesh structure in all Ppy concentrationstested, and displayed electrical conductivities in the range of nativecardiac tissue. Furthermore, these materials exhibit tensile strength,surface roughness, and wettability values appropriate for their finaluse as cardiac patches. In vitro experiments withcardiac fibroblasts and H9c2 cells confirmed the exceptional biocompatibilityof BC-Ppy composites. BC-Ppy scaffolds improved cell viability andattachment, promoting a desirable cardiomyoblast morphology. Biochemicalanalyses revealed that H9c2 cells showed different cardiomyocyte phenotypesand distinct levels of maturity depending on the amount of Ppy inthe substrate used. Specifically, the employment of BC-Ppy compositesdrives partial H9c2 differentiation toward a cardiomyocyte-like phenotype.The scaffolds increase the expression of functional cardiac markersin H9c2 cells, indicative of a higher differentiation efficiency,which is not observed with plain BC. Our results highlight the remarkablepotential use of BC-Ppy scaffolds as a cardiac patch in tissue regenerativetherapies.

JTD Keywords: bacterial nanocellulose, cardiac patches, conducting polymers, polypyrrole, Arrhythmias, Bacterial nanocellulose, Biomaterials, Cardiac patches, Cell therapy, Cellulose, Conductingpolymers, H9c2, In-vitro, Polymer, Polypyrrole, Scaffolds, Tissue, Tissue engineering, Viability

JTD Keywords: alpha-tricalcium phosphate, cement, crystallinity, hydrolysis, hydroxyapatite, particle-size, powders, relative humidity, setting reaction, stability, tcp, vapor sorption, Calcium-phosphate, Moisture

Polymeric spherical nucleic acids comprise pH-sensitive, polymer-conjugated oligonucleotides that self-assemble into nanoparticles with the ability to escape endosomes, overcoming a major obstacle in nucleic acid delivery.

JTD Keywords: c9orf72, cellular uptake, dna, encapsulation, expansion, ph, stability, trafficking, vesicles, Hexanucleotide repeat

Despite the high homology of OmpF and OmpC, the internally folded loop responds differently to temperature increase.

JTD Keywords: diffusion, mechanism, molecules, nanopores, permeability, proteins, rules, simulations, transport, Membrane

Microfluidics represents a very promising technological solution for conducting massive biological experiments. However, the difficulty of managing the amount of information available often precludes the wide potential offered. Using machine learning, we aim to accelerate microfluidics uptake and lead to quantitative and reliable findings. In this work, we propose complementing microfluidics with machine learning (MLM) approaches to enhance the diagnostic capability of lab-on-chip devices. The introduction of data analysis methodologies within the deep learning framework corroborates the possibility of encoding cell morphology beyond the standard cell appearance. The proposed MLM platform is used in a diagnostic test for blood diseases in murine RBC samples in a dedicated microfluidics device in flow. The lack of plasticity of RBCs in Pyruvate Kinase Disease (PKD) is measured massively by recognizing the shape deformation in RBCs walking in a forest of pillars within the chip. Very high accuracy results, far over 85 %, in recognizing PKD from control RBCs either in simulated and in real experiments demonstrate the effectiveness of the platform.

JTD Keywords: Blood disease, Deep transfer learning, Deficiency, Deformability, Machine learning microfluidics, Video analysis

(1) Background: Restoring arm and hand function is a priority for individuals with cervical spinal cord injury (cSCI) for independence and quality of life. Transcutaneous spinal cord stimulation (tSCS) promotes the upper extremity (UE) motor function when applied at the cervical region. The aim of the study was to determine the effects of cervical tSCS, combined with an exoskeleton, on motor strength and functionality of UE in subjects with cSCI. (2) Methods: twenty-two subjects participated in the randomized mix of parallel-group and crossover clinical trial, consisting of an intervention group (n = 15; tSCS exoskeleton) and a control group (n = 14; exoskeleton). The assessment was carried out at baseline, after the last session, and two weeks after the last session. We assessed graded redefined assessment of strength, sensibility, and prehension (GRASSP), box and block test (BBT), spinal cord independence measure III (SCIM-III), maximal voluntary contraction (MVC), ASIA impairment scale (AIS), and WhoQol-Bref; (3) Results: GRASSP, BBT, SCIM III, cylindrical grip force and AIS motor score showed significant improvement in both groups (p ≤ 0.05), however, it was significantly higher in the intervention group than the control group for GRASSP strength, and GRASSP prehension ability (p ≤ 0.05); (4) Conclusion: our findings show potential advantages of the combination of cervical tSCS with an exoskeleton to optimize the outcome for UE.

JTD Keywords: arm function, cervical spinal cord injury, electrical-stimulation, functional walking, functionality, grip force, hand function, individuals, injury, motor function, reliability, robotics, spasticity, transcutaneous electrical spinal cord stimulation, upper extremity, Epidural stimulation, Transcutaneous electrical spinal cord stimulation, Upper extremity

Mechanical force modulates the conformation and function of individual proteins, and this underpins many mechanically driven cellular processes. This Review addresses single-molecule force spectroscopy experiments conducted on proteins with a known role in mechanosensing and mechanotransduction in eukaryotic cells.; In addition to biochemical signals and genetic considerations, mechanical forces are rapidly emerging as a master regulator of human physiology. However, the molecular mechanisms that regulate force-induced functionalities across a wide range of scales, encompassing the cell, tissue or organ levels, are not well understood in comparison. With the advent, development and refining of single-molecule nanomechanical techniques that enable the conformational dynamics of individual proteins under the effect of a calibrated force to be probed, we have begun to acquire a comprehensive knowledge of the diverse physicochemical principles that regulate the elasticity of single proteins. Here, we review the major advances underpinning our current understanding of how the elasticity of single proteins regulates mechanosensing and mechanotransduction. We discuss the present limitations and future challenges of this prolific and burgeoning field.

JTD Keywords: Cadherin adhesion, Energy landscape, Extracellular-matrix protein, Focal adhesion kinase, Mechanical stability, Molecule force spectroscopy, Muscle protein, N2b element, Stranded-dna, Structural basis

Breathing pattern has been shown to be different in chronic obstructive pulmonary disease (COPD) patients compared to healthy controls during rest and walking. In this study we evaluated respiratory parameters and the breathing variability of COPD patients as a function of their severity. Thoracic bioimpedance was acquired on 66 COPD patients during the performance of the six-minute walk test (6MWT), as well as 5 minutes before and after the test while the patients were seated, i.e. resting and recovery phases. The patients were classified by their level of airflow limitation into moderate and severe groups. We characterized the breathing patterns by evaluating common respiratory parameters using only wearable bioimpedance. Specifically, we computed the median and the coefficient of variation of the parameters during the three phases of the protocol, and evaluated the statistical differences between the two COPD severity groups. We observed significant differences between the COPD severity groups only during the sitting phases, whereas the behavior during the 6MWT was similar. Particularly, we observed an inverse relationship between breathing pattern variability and COPD severity, which may indicate that the most severely diseased patients had a more restricted breathing compared to the moderate patients.

JTD Keywords: 6mwt, activation, breathing pattern, burden, chronic obstructive pulmonary disease, exercise, muscles, pressure, pulmonary, signals, variability, volumes, wearables, Bioimpedance, Impedance pneumography

Building functional mimics of cell membranes is an important task toward the development of synthetic cells. So far, lipid and amphiphilic block copolymers are the most widely used amphiphiles with the bilayers by the former lacking stability while membranes by the latter are typically characterized by very slow dynamics. Herein, we introduce a new type of Janus dendrimer containing a zwitterionic phosphocholine hydrophilic headgroup (JDPC ) and a 3,5-substituted dihydrobenzoate-based hydrophobic dendron. JDPC self-assembles in water into zwitterionic dendrimersomes (z-DSs) that faithfully recapitulate the cell membrane in thickness, flexibility, and fluidity, while being resilient to harsh conditions and displaying faster pore closing dynamics in the event of membrane rupture. This enables the fabrication of hybrid DSs with components of natural membranes, including pore-forming peptides, structure-directing lipids, and glycans to create raft-like domains or onion vesicles. Moreover, z-DSs can be used to create active synthetic cells with life-like features that mimic vesicle fusion and motility as well as environmental sensing. Despite their fully synthetic nature, z-DSs are minimal cell mimics that can integrate and interact with living matter with the programmability to imitate life-like features and beyond. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

JTD Keywords: biological-membranes, bottom-up synthetic biology, chain, hybrid vesicles, hydroethidine, organization, polymersome, proteins, stability, synthetic cells, thickness, vesicle fusion, vesicle motility, vesicles, zwitterionic dendrimersomes, Biosensor, Biosensors, Bottom-up synthetic biology, Hybrid vesicles, Lipid-bilayers, Synthetic cells, Vesicle fusion, Vesicle motility, Zwitterionic dendrimersomes

Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.

JTD Keywords: Bioceramics, Bone, Bone regeneration, Composites, Degradation, Fabrication, Hydroxyapatite, Hydroxyapatite scaffolds, Injectability, Porosity, Recombinant spider silk, Rgd motifs, Silk, Stem-cells

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: 3d architectures, biomolecular gradients, colon, crypt, engineering organoids, hydrogels, identification, in silico modeling, intestinal stem cell niches, light sheet fluorescence microscopy, niche, permeability, photolithography, regeneration, villus, wnt, 3d architectures, Biomolecular gradients, Engineering organoids, In silico modeling, Intestinal stem cell niches, Light sheet fluorescence microscopy, Photolithography, Stem-cell

Hydrogels have been recently proposed as suitable materials to generate reactive oxygen and nitrogen species (RONS) upon gas-plasma treatment, and postulated as promising alternatives to conventional cancer therapies. Acting as delivery vehicles that allow a controlled release of RONS to the diseased site, plasma-treated hydrogels can overcome some of the limitations presented by plasma-treated liquids in in vivo therapies. In this work, we optimized the composition of a methylcellulose (MC) hydrogel to confer it with the ability to form a gel at physiological temperatures while remaining in the liquid phase at room temperature to allow gas-plasma treatment with suitable formation of plasma-generated RONS. MC hydrogels demonstrated the capacity for generation, prolonged storage and release of RONS. This release induced cytotoxic effects on the osteosarcoma cancer cell line MG-63, reducing its cell viability in a dose-response manner. These promising results postulate plasma-treated thermosensitive hydrogels as good candidates to provide local anticancer therapies.

JTD Keywords: Case-control studies, Cellulose, Hydrogels, Methylcellulose, Phase-separation, Plasma gases, Reactive oxygen species, Stability, Substituent, Temperature, Thermoreversible gelation

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(?-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.

JTD Keywords: cancer nanomedicine, cell trafficking, delivery, direct stochastic optical reconstruction microscopy (dstorm), nanoparticle stability, poly(beta-aminoester) nanoparticles, Direct stochastic optical reconstruction microscopy (dstorm), Poly(?-aminoester) nanoparticles, Poly(beta-amino ester)s, Poly(β-aminoester) nanoparticles

Effective inhibition of sperm motility using a spermicide can be a promising approach in developing non-invasive male contraceptive agents. Copper is known to have contraceptive properties and has been used clinically for decades as intrauterine contraceptive devices (IUDs) for contraception in females. Beyond that, the spermicidal use of copper is not explored much further, even though its use can also subdue the harmful effects caused by the hormonal female contraceptive agents on the environment. Herein, the size, concentration, and time-dependent in vitro inhibition of bovine spermatozoa by copper microparticles are studied. The effectivity in inhibiting sperm motility is correlated with the amount of Cu2+ ions released by the particles during incubation. The copper particles cause direct suppression of sperm motility and viability upon incubation and thereby show potential as sperm-inhibiting, hormone-free candidate for male contraception. In addition, biocompatibility tests using a cervical cell line help optimizing the size and concentration of the copper particles for the best spermicidal action while avoiding toxicity to the surrounding tissue.

JTD Keywords: Bovine spermatozoa, Clinical-trial, Copper, Human-spermatozoa, Ions, Male contraception, Metallic copper, Microparticles, Progestins, Sperm motility, Sperm viability, Spermicide, Viability

We investigated the diffusion of three cyclic boronates formulated as beta-lactamase inhibitors through the porin OmpF to evaluate their potential to cross OM via the porin pathway. The three nonbeta-lactam molecules diffuse through the porin eyelet region with the same mechanism observed for beta-lactam molecules and diazobicyclooctan derivatives, with the electric dipole moment aligned with the transversal electric field. In particular, the BOH group can interact with both the basic ladder and the acidic loop L3, which is characteristic of the size-constricted region of this class of porins. On one hand, we confirm that the transport of small molecules through enterobacter porins has a common general mechanism; on the other, the class of cyclic boronate molecules does not seem to have particular difficulties in diffusing through enterobacter porins, thus representing a good scaffold for new anti-infectives targeting Gram-negative bacteria research.

JTD Keywords: beta-lactamase inhibitors, cyclic boronates, diffusion current, metadynamics, molecular dynamics simulations, permeation, Antibiotics, Beta-lactamase inhibitors, Cyclic boronates, Diffusion, Diffusion current, Discovery, Electric-field, Metadynamics, Molecular dynamics simulations, Molecular-dynamics simulations, Nanopores, Permeability, Permeation, Porins, Rules, Translocation

Tuning of the crosslink density (CLD) in the rubber compounds is very crucial for optimizing the physical and mechanical properties of the ultimate rubber products. Conventionally, CLD can be measured via rheological methods such as moving die rheometer (MDR), via mechanical tests such as temperature scanning stress relaxation analysis (TSSR), or via direct swelling experiments using Flory–Rehner approach. In the current study, two novel techniques, focused ion beam - scanning electron microscopy (FIB-SEM) processing, with simultaneous energy dispersive X-ray spectrometry (EDS) mapping analysis and scanning acoustic microscopy (SAM) were combined and correlated to conventional methods on a model recipe of ethylene propylene diene monomer (EPDM) compound having different sulphur contents. Depending on the applied technique, the increase in the crosslink density with sulphur content was found to be 1.7 fold for the Flory–Rehner approach and 1.2 fold for both TSSR and MDR. It is directly monitored from the FIB-SEM-EDS analysis that the sulphur distribution and agglomeration behavior increased in line with ZnO content, which is an indirect indication of the rise in crosslink density. The impedance maps of the crosslinked samples obtained through SAM analysis revealed that the impedance of the samples increased with the increasing sulphur content, which can be attributed to higher level of crosslink density. A quantified correlation was obtained between SAM images and the crosslink density of the samples. It was shown that SAM is a promising tool for practical and non-destructive analysis for determining the formation of crosslink density of the rubbers. © The Author(s) 2022.

JTD Keywords: blends, compressibility, crosslink density, cure characteristics, ethylene propylene diene monomer, focused ion beam, mechanical-properties, morphology, natural-rubber, particles, scanning acoustic microscopy, scanning electron microscopy, sulfur, thermal-stability, vulcanization, Composite soft materials, Cross-link densities, Crosslink density, Crosslinking, Density (specific gravity), Ethylene, Ethylene propylene diene monomer, Flory-rehner, Focused ion beam - scanning electron microscopy, Focused ion beam-scanning electron microscopies, Ii-vi semiconductors, Monomers, Moving die rheometers, Physical and mechanical properties, Propylene, Relaxation analysis, Rubber, Scanning acoustic microscopy, Scanning electron microscopy, Stress relaxation, Sulfur contents, Temperature scanning stress relaxations, Zinc oxide

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Such forces can lead to the nuclear translocation of proteins, but whether force controls nucleocytoplasmic transport, and how, remains unknown. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than for facilitated diffusion. Owing to this differential effect, force leads to the translocation of cargoes into or out of the nucleus within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism and engineered exogenously by introducing appropriate nuclear localization signals. Our work unveils a mechanism of mechanically induced signalling, probably operating in parallel with others, with potential applicability across signalling pathways.; Andreu et al. show that force regulates nucleocytoplasmic transport by weakening the permeability barrier of nuclear pore complexes, affecting passive and facilitated diffusion in different ways.

JTD Keywords: Activation, Inhibitor, Matrix, Mechanotransduction, Nesprins, Nucleoporins, Permeability barrier, Pore complex, Proteins, Transmission

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

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

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid were used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conducted on the nanopatterns. Cohesion and GJIC were evaluated in cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

JTD Keywords: arginine-glycine-aspartic acid, arginine–glycine–aspartic acid, cell adhesion, condensation, dendrimer-based nanopatterning, gap junction intercellular communication, Actin, Adhesion, Arginine-glycine-aspartic acid, Cell adhesion, Collagen, Condensation, Connexin-43, Dendrimer-based nanopatterning, Dynamics, Extracellular-matrix, Fibronectin, Gap junction intercellular communication, Mesenchymal stem cells, Permeability, Phenotype, Vinculin

Objective: To investigate the effects of single-session premotor and primary motor tDCS in chronic stroke patients with relation to possible inter-hemispheric interactions. Methods: Anodal tDCS of either M1 or premotor cortex of the side contralateral to the paretic hand, cathodal tDCS of the premotor cortex of the side ipsilateral to the paretic hand and sham stimulation were performed in 12 chronic stroke patients with mild hand paresis in a balanced cross-over design. The Jebsen-Taylor Hand Function test, evaluating the time required for performance of everyday motor tasks, was employed. Results: The repeated-measure ANOVA with Greenhouse-Geisser correction showed significant influence of the stimulation type (factor SESSION; F(2.6, 28.4) = 47.3, p < 0.001), the test performance time relative to stimulation (during or after tDCS; factor TIME, F(1.0, 11.0) = 234.5, p < 0.001) with higher effect after the stimulation and the interaction SESSION*TIME (F(1.7, 1.2) = 30.5, p < 0.001). All active conditions were effective for the modulation of JTT performance, though the highest effect was observed after anodal tDCS of M1, followed by effects after anodal stimulation of the premotor cortex contralateral to the paretic hand. Based on the correlation patterns, the inhibitory input to M1 from premotor cortex of another hemisphere and an excitatory input from the ipsilesional premotor cortex were suggested. Conclusion: The premotor cortex is a promising candidate area for transcranial non-invasive stimulation of chronic stroke patients. © 2022 The Author(s)

JTD Keywords: areas, contralateral primary motor, dorsal premotor, excitability, jtt, lateral premotor, object manipulation, premotor cortex, recovery, stroke, tdcs, time-course, transcranial direct current stimulation, Jtt, Noninvasive brain-stimulation, Premotor cortex, Stroke, Tdcs, Transcranial direct current stimulation

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

Micro- and nanotexturization of surfaces can give to the parts different advanced functionalities, such as superhydrophobicity, self-cleaning, or antibacterial capabilities. These advanced properties in combination with the biocompatibility of Liquid Silicone Rubber are an interesting approach for obtaining high-performance medical devices. The industrial production of surface textures in polymeric materials is through the replication technique, and the best option to attain a high production rate is injection molding. Moreover, its low viscosity during processing can provide an accurate replication capacity by the easy filling by capillarity of the microtextures. An innovative replicating technique for Liquid Silicone Rubber is presented by studying the replication of different shaped textures within a diameter range of between 2 and 50 mu m. The copying process consists in the overmolding of a textured polymeric inlay obtained by nanoimprint lithography. At the end of the process, a textured part is obtained, while the imprinted film remains in the mold. The injection molding parameters are optimized to increase the replication accuracy, and their effect on texture replicability is analyzed and discussed. Finally, it is shown that the textured surfaces improve their wettability behavior, which is a necessary and important characteristic in the development of biomedical devices.

JTD Keywords: Cross-linking density, Injection molding, Microtextures, Nanoimprint lithography, Polymeric inlays, Silicone rubber, Stamp, Wettability

Underpowered trials risk inaccurate results. Recruitment to stroke rehabilitation randomised controlled trials (RCTs) is often a challenge. Statistical simulations offer an important opportunity to explore the adequacy of sample sizes in the context of specific outcome measures. We aimed to examine and compare the adequacy of stroke rehabilitation RCT sample sizes using the Barthel Index (BI) or modified Rankin Scale (mRS) as primary outcomes.We conducted computer simulations using typical experimental event rates (EER) and control event rates (CER) based on individual participant data (IPD) from stroke rehabilitation RCTs. Event rates are the proportion of participants who experienced clinically relevant improvements in the RCT experimental and control groups. We examined minimum sample size requirements and estimated the number of participants required to achieve a number needed to treat within clinically acceptable boundaries for the BI and mRS.We secured 2350 IPD (18 RCTs). For a 90% chance of statistical accuracy on the BI a rehabilitation RCT would require 273 participants per randomised group. Accurate interpretation of effect sizes would require 1000s of participants per group. Simulations for the mRS were not possible as a clinically relevant improvement was not detected when using this outcome measure.Stroke rehabilitation RCTs with large sample sizes are required for accurate interpretation of effect sizes based on the BI. The mRS lacked sensitivity to detect change and thus may be unsuitable as a primary outcome in stroke rehabilitation trials.Copyright © 2021 Elsevier Inc. All rights reserved.

JTD Keywords:  , barthel index, design, increasing value, modified rankin scale, randomised controlled trials, recruitment, reducing waste, reliability, sample size calculations, simulations, stroke rehabilitation, Adult, Article, Barthel index, Calculation, Computer simulation, Controlled study, Effect size, Female, Human, Human experiment, Major clinical study, Male, Modified rankin scale, Numbers needed to treat, Outcome assessment, Randomised controlled trials, Randomized controlled trial, Randomized controlled-trials, Rankin scale, Recruitment, Rehabilitation, Sample size, Sample size calculations, Simulations, Stroke rehabilitation

Maintaining a balance between excitatory and inhibitory activity is an essential feature of neural networks of the neocortex. In the face of perturbations in the levels of excitation to cortical neurons, synapses adjust to maintain excitatory-inhibitory (EI) balance. In this review, we summarize research on this EI homeostasis in the neocortex, using stroke as our case study, and in particular the loss of excitation to distant cortical regions after focal lesions. Widespread changes following a localized lesion, a phenomenon known as diaschisis, are not only related to excitability, but also observed with respect to functional connectivity. Here, we highlight the main findings regarding the evolution of excitability and functional cortical networks during the process of post-stroke recovery, and how both are related to functional recovery. We show that cortical reorganization at a global scale can be explained from the perspective of EI homeostasis. Indeed, recovery of functional networks is paralleled by increases in excitability across the cortex. These adaptive changes likely result from plasticity mechanisms such as synaptic scaling and are linked to EI homeostasis, providing a possible target for future therapeutic strategies in the process of rehabilitation. In addition, we address the difficulty of simultaneously studying these multiscale processes by presenting recent advances in large-scale modeling of the human cortex in the contexts of stroke and EI homeostasis, suggesting computational modeling as a powerful tool to tie the meso- and macro-scale processes of recovery in stroke patients. Copyright © 2022 Páscoa dos Santos and Verschure.

JTD Keywords: balanced excitation, canonical microcircuit, cerebral-cortex, cortical excitability, cortical reorganization, diaschisis, excitability, excitatory-inhibitory balance, functional networks, homeostatic plasticity, ischemic-stroke, neuronal avalanches, photothrombotic lesions, state functional connectivity, whole-brain models, Algorithm, Biological marker, Brain, Brain cell, Brain cortex, Brain function, Brain radiography, Cerebrovascular accident, Cortical reorganization, Diaschisis, Down regulation, Excitability, Excitatory-inhibitory balance, Fluorine magnetic resonance imaging, Functional networks, Homeostasis, Homeostatic plasticity, Human, Motor dysfunction, Neuromodulation, Plasticity, Pyramidal nerve cell, Review, Simulation, Stroke, Stroke patient, Theta-burst stimulation, Visual cortex

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

Drug development is an ever-growing field, increasingly requesting reliable in vitro tools to speed up early screening phases, reducing the need for animal experiments. In oral delivery, understanding the absorption pattern of a new drug in the small intestine is paramount. Classical two-dimensional (2D) in vitro models are generally too simplistic and do not accurately represent native tissues. The main goal of this work was to develop an advanced three-dimensional (3D) in vitro intestinal model to test absorption in a more reliable manner, by better mimicking the native environment. The 3D model is composed of a collagen-based stromal layer with embedded fibroblasts mimicking the intestinal lamina propria and providing support for the epithelium, composed of enterocytes and mucus-secreting cells. An endothelial layer, surrogating the absorptive capillary network, is also present. The cellular crosstalk between the different cells present in the model is unveiled, disclosing key players, namely those involved in the contraction of collagen by fibroblasts. The developed 3D model presents lower levels of P-glycoprotein (P-gp) and Multidrug Resistance Protein 2 (MRP2) efflux transporters, which are normally overexpressed in traditional Caco-2 models, and are paramount in the absorption of many compounds. This, allied with transepithelial electrical resistance (TEER) values closer to physiological ranges, leads to improved and more reliable permeability outcomes, which are observed when comparing our results with in vivo data.

JTD Keywords: 3d intestinal model, drug absorption, drug development, endothelium, hydrogel, 3d intestinal model, 3d modeling, 3d models, 3d-modeling, Alkaline-phosphatase, Animal experiments, Biopharmaceutics classification, Caco-2 cells, Cell culture, Collagen, Collagen gel, Drug absorption, Drug development, Endothelium, Fibroblasts, Glycoproteins, Hydrogel, In-vitro, Matrix metalloproteinases, Membrane-permeability, Paracellular transport, Permeability, Single-pass vs., Speed up

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

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

Polypeptide-based nanoparticles offer unique advantages from a nanomedicine perspective such as biocompatibility, biodegradability, and stimuli-responsive properties to (patho)physiological conditions. Conventionally, self-assembled polypeptide nanostructures are prepared by first synthesizing their constituent amphiphilic polypeptides followed by postpolymerization self-assembly. Herein, we describe the one-pot synthesis of oxidation-sensitive supramolecular micelles and vesicles. This was achieved by polymerization-induced self-assembly (PISA) of the N-carboxyanhydride (NCA) precursor of methionine using poly(ethylene oxide) as a stabilizing and hydrophilic block in dimethyl sulfoxide (DMSO). By adjusting the hydrophobic block length and concentration, we obtained a range of morphologies from spherical to wormlike micelles, to vesicles. Remarkably, the secondary structure of polypeptides greatly influenced the final morphology of the assemblies. Surprisingly, wormlike micellar morphologies were obtained for a wide range of methionine block lengths and solid contents, with spherical micelles restricted to very short hydrophobic lengths. Wormlike micelles further assembled into oxidation-sensitive, self-standing gels in the reaction pot. Both vesicles and wormlike micelles obtained using this method demonstrated to degrade under controlled oxidant conditions, which would expand their biomedical applications such as in sustained drug release or as cellular scaffolds in tissue engineering.

JTD Keywords: alpha-amino-acid, hydrogels, leuchs anhydrides, platform, polypeptides, transformation, triggered cargo release, Amino acids, Amphiphilics, Biocompatibility, Biodegradability, Block lengths, Controlled drug delivery, Dimethyl sulfoxide, Ethylene, Gels, Hydrophobicity, Medical nanotechnology, Methionine, Micelles, Morphology, Nanoparticles, One-pot synthesis, Organic solvents, Oxidation, Physiological condition, Polyethylene glycols, Polyethylene oxides, Polymerization, Post-polymerization, Ring-opening polymerization, Scaffolds (biology), Self assembly, Stimuli-responsive properties, Supramolecular chemistry, Supramolecular gels, Supramolecular micelles, Wormlike micelle

Patients suffering from obstructive sleep apnea (OSA) usually present an increased sympathetic activity caused by the intermittent hypoxia effect on autonomic control. This study evaluated the relationship between sleep stages and the apnea duration, frequency, and type, as well as their impact on HRV markers in different groups of disease severity. The hypnogram and R-R interval signals were extracted in 81 OSA patients from night polysomnographic (PSG) recordings. The apnea-hypopnea index (AHI) defined patient classification as mild-moderate (AHI< 30, n 44) or severe (AHI>30, n 37). The normalized power in VLH, LF, and HF bands of RR series were estimated by a time-frequency approach and averaged in 1-min epochs of normal and apnea segments. The autonomic response and the impact of sleep stages were assessed in both segments to compare patient groups. Deeper sleep stages (particularly S2) concentrated the shorter and mild apnea episodes (from 10 to 40 s) compared to light (SWS) and REM sleep. Longer episodes (>50 s) although less frequent, were of similar incidence in all stages. This pattern was more pronounced for the group of severe patients. Moreover, during apnea segments, LF nu was higher (p 0.044) for the severe group, since V LF nu and HF nu presented the greatest changes when compared to normal segments. The non-REM sleep seems to better differentiate OSA patients groups, particularly through VLF nu and HF nu (p<0.001). A significant difference in both sympathetic and vagal modulation between REM and non-REM sleep was only found within the severe group. These results confirm the importance of considering sleep stages for HRV analysis to further assess OSA disease severity, beyond the traditional and clinically limited AHI values.Clinical relevance - Accounting for sleep stages during HRV analysis could better assess disease severity in OSA patients. © 2021 IEEE.

JTD Keywords: blood-pressure, genomic consequences, intermittent hypoxia, rapid-eye-movement, sympathetic activity, Heart rate, Heart-rate-variability, Human, Humans, Polysomnography, Rem sleep, Sleep apnea, obstructive, Sleep disordered breathing, Sleep stage, Sleep stages, Sleep, rem

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions.© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

JTD Keywords: dc-sign, density, dimerization, endocytosis, lateral mobility, ligand-binding, mannose receptor, proteins, recognition, Animal, Animals, Cell membrane, Cell membrane permeability, Chemistry, Cho cell line, Cho cells, Cricetulus, Cysteine-rich domain, Kinetics, Lectin, Lectins, Ligand, Ligands, Molecular library, Multivariate analysis, Polysaccharide, Polysaccharides, Procedures, Protein binding, Single molecule imaging, Small molecule libraries, Structure activity relation, Structure-activity relationship

3D printing is a competitive manufacturing technology, which has opened up new possibilities for the fabrication of complex ceramic structures and customised parts. Extrusion-based technologies, also known as direct ink writing (DIW) or robocasting, are amongst the most used for ceramic materials. In them, the rheological properties of the ink play a crucial role, determining both the extrudability of the paste and the shape fidelity of the printed parts. However, comprehensive rheological studies of printable ceramic inks are scarce and may be difficult to understand for non-specialists. The aim of this review is to provide an overview of the main types of ceramic ink formulations developed for DIW and a detailed description of the more relevant rheological tests for assessing the printability of ceramic pastes. Moreover, the key rheological parameters are identified and linked to printability aspects, including the values reported in the literature for different ink compositions.

JTD Keywords: 3-dimensional structures, behavior, deposition, direct ink writing, freeform fabrication, gelation, glass scaffolds, mechanical-properties, printability, rheology, robocasting, suspensions, 3d printing, Direct ink writing, Phosphate scaffolds, Printability, Rheology, Robocasting

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

JTD Keywords: cell, electrochemical microscopy, membrane-permeability, microphysiological systems, organs-chips, platform, scanning electrochemical microscopy, scanning ion conductance microscopy, scanning probe microscopy, shear-stress, surface-topography, Ion conductance microscopy, Microphysiological systems, Organs-chips, Scanning electrochemical microscopy, Scanning ion conductance microscopy, Scanning probe microscopy

The extracellular matrix mechanical properties regulate processes in development, cancer, and fibrosis. Among the distinct mechanical properties, the vast majority of research has focused on the extracellular matrix's elasticity as the primary determinant of cell and tissue behavior. However, both cells and the extracellular matrix are not only elastic but also viscous. Despite viscoelasticity being a universal feature of living tissues, our knowledge of the influence of the extracellular matrix's viscoelasticity in cell behavior is limited. This mini-review describes some of the recent findings that have highlighted the role of the extracellular matrix's viscoelasticity in cell and tissue dynamics.

JTD Keywords: behavior, cell adhesion, cell mechanics, cell migration, deformability, extracellular matrix, extracellular matrix mechanics, fluidity, forces, hydrogels, mechanobiology, mechanotransduction, tissue mechanics, viscoelasticity, viscosity, Cell adhesion, Cell mechanics, Cell migration, Elasticity, Extracellular matrix, Extracellular matrix mechanics, Fluidity, Mechanobiology, Mechanotransduction, Migration, Tissue mechanics, Viscoelasticity, Viscosity

A plethora of applications using polysaccharides have been developed in recent years due to their availability as well as their frequent nontoxicity and biodegradability. These polymers are usually obtained from renewable sources or are byproducts of industrial processes, thus, their use is collaborative in waste management and shows promise for an enhanced sustainable circular economy. Regarding the development of novel delivery systems for biotherapeutics, the potential of polysaccharides is attractive for the previously mentioned properties and also for the possibility of chemical modification of their structures, their ability to form matrixes of diverse architectures and mechanical properties, as well as for their ability to maintain bioactivity following incorporation of the biomolecules into the matrix. Biotherapeutics, such as proteins, growth factors, gene vectors, enzymes, hormones, DNA/RNA, and antibodies are currently in use as major therapeutics in a wide range of pathologies. In the present review, we summarize recent progress in the development of polysaccharide-based hydrogels of diverse nature, alone or in combination with other polymers or drug delivery systems, which have been implemented in the delivery of biotherapeutics in the pharmaceutical and biomedical fields. © 2021 American Chemical Society.

JTD Keywords: biodegradable dextran hydrogels, biotherapeutics, bone morphogenetic protein-2, carrageenan-based hydrogels, chitosan-based hydrogels, controlled delivery, controlled-release, cross-linked hydrogels, growth-factor delivery, hydrogels, in-vitro characterization, polysaccharides, self-healing hydrogel, stimuli-responsiveness, tissue engineering, Antibodies, Bioactivity, Biodegradability, Biomedical fields, Biomolecules, Biotherapeutics, Chemical modification, Circular economy, Controlled delivery, Controlled drug delivery, Delivery systems, Drug delivery system, Functional polymers, Hyaluronic-acid hydrogels, Hydrogels, Industrial processs, Polysaccharides, Recent progress, Renewable sources, Stimuli-responsiveness, Targeted drug delivery, Tissue engineering, Waste management

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ⁓1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

JTD Keywords: amplitude-modulation am-afm, atomic-force microscopy, capillary, force reconstruction, multifrequency afm, nanoscale wetting, persistence, reconstruction, relative-humidity, surfaces, tobacco mosaic virus (tmv), tobamovirus, transmission, water, Amplitude-modulation am-afm, Force reconstruction, Humidity, Microscopy, atomic force, Multifrequency afm, Nanoscale wetting, Tobacco mosaic virus, Tobacco mosaic virus (tmv), Tobacco mosaic virus (tmv), nanoscale wetting, Tobacco-mosaic-virus, Virion, Water, Wettability

Objective. Impaired trunk stability is frequent in spinal cord injury (SCI), but there is a lack of quantitative measures for assessing trunk function. Our objectives were to: (a) evaluate trunk muscle activity and movement patterns during a reaching task in SCI patients, (b) compare the impact of cervical (cSCI) and thoracic (tSCI) injuries in trunk function, and (c) investigate the effects of a startling acoustic stimulus (SAS) in these patients. Approach. Electromyographic (EMG) and smartphone accelerometer data were recorded from 15 cSCI patients, nine tSCI patients, and 24 healthy controls, during a reaching task requiring trunk tilting. We calculated the response time (RespT) until pressing a target button, EMG onset latencies and amplitudes, and trunk tilt, lateral deviation, and other movement features from accelerometry. Statistical analysis was applied to analyze the effects of group (cSCI, tSCI, control) and condition (SAS, non-SAS) in each outcome measure. Main results. SCI patients, especially those with cSCI, presented significantly longer RespT and EMG onset latencies than controls. Moreover, in SCI patients, forward trunk tilt was accompanied by significant lateral deviation. RespT and EMG latencies were remarkably shortened by the SAS (the so-called StartReact effect) in tSCI patients and controls, but not in cSCI patients, who also showed higher variability. Significance. The combination of EMG and smartphone accelerometer data can provide quantitative measures for the assessment of trunk function in SCI. Our results show deficits in postural control and compensatory strategies employed by SCI patients, including delayed responses and higher lateral deviations, possibly to improve sitting balance. This is the first study investigating the StartReact responses in trunk muscles in SCI patients and shows that the SAS significantly accelerates RespT in tSCI, but not in cSCI, suggesting an increased cortical control exerted by these patients.

JTD Keywords: accelerometer, electromyography, impairment, individuals, movements, postural stability, reaction-time, reliability, sitting balance, smartphone, spinal cord injury, startle, startreact, strategies, stroke, trunk, Accelerometer, Electromyography, Sitting balance, Smartphone, Spinal cord injury, Startreact, Trunk

A new methodology for the pH-triggered degradation of polymers or for the release of drugs under visible light irradiation based on the cyclization of ortho-hydroxy-cinnamates (oHC) to coumarins is described. The key oHC structural motif can be readily incorporated into the rational design of novel photocleavable polymers via click chemistry. This main-chain moiety undergoes a fast photocleavage when irradiated with 455 nm light provided that a suitable base is added. A series of polyethylene glycol-alt-ortho-hydroxy cinnamate (polyethylene glycol (PEG)(n)-alt-oHC)-based polymers are synthesized and the time-dependent visible-light initiated cleavage of the photoactive monomer and polymer is investigated in solution by a variety of spectroscopic and chromatographic techniques. The photo-degradation behavior of the water-soluble poly(PEG(2000)-alt-oHC) is investigated within a broad pH range (pH = 2.1-11.8), demonstrating fast degradation at pH 11.8, while the stability of the polymer is greatly enhanced at pH 2.1. Moreover, the neat polymer shows long-term stability under daylight conditions, thus allowing its storage without special precautions. In addition, two water-soluble PEG-based drug-carrier molecules (mPEG(2000)-oHC-benzhydrol/phenol) are synthesized and used for drug delivery studies, monitoring the process by UV-vis spectroscopy in an ON/OFF intermittent manner.

JTD Keywords: coumarins, drug delivery, e/z-double bond isomerization, o-hydroxy cinnamates, polymer degradation, Aliphatic compounds, Antioxidant activity, Antitumor, Chromatographic techniques, Chromatography, Cis/trans isomerization, Controlled drug delivery, Coumarin derivatives, Coumarins, Drug delivery, Drug delivery system, E/z-double bond isomerization, Films, Hydrogels, Image enhancement, Light, Long term stability, O-hydroxy cinnamates, Particles, Photoactive monomers, Photodegradation, Polyethylene glycols, Polyethylenes, Polymer degradation, Responsive polymers, Salts, Structural motifs, Synthesis (chemical), Targeted drug delivery, Visible light photocatalysis, Visible-light irradiation

© 2021 Wiley-VCH GmbH One limitation of wearable electronics, and at the same time a challenge, is the lack of energy storage devices with multiple functionalities produced using clean and environmental-friendly strategies. Here, a multifunctional conductive hydrogel containing poly(3,4-ethylenedioxythiophene) (PEDOT) and alginate is fabricated, to be used as electrodes in supercapacitors, by applying water-mediated self-assembly and polymerization processes at room temperature. The interpenetration of both polymers allows the combination of flexibility and self-healing properties within the same hydrogel together with the intrinsic biocompatibility and sustainability of such materials. Initially, PEDOT:polystyrene sulfonate and alginate aqueous solutions are mixed in two different proportions (1:1 and 1:3) and ionically crosslinked with CaCl2. Subsequently, re-interpenetration of poly(hydroxymethyl-3,4-ethylenedioxythiophene) by anodic polymerization in CaCl2 aqueous solution is achieved. Re-interpenetrated 1:3 PEDOT/alginate hydrogels show excellent capacitance values (35 mF cm−2) and good capacitance retention. In addition, the electrochemical properties are not significantly changed after many cutting/self-healing cycles as observed by cyclic voltammetry. Therefore, this sustainably produced hydrogel shows promising properties for use in wearable energy storage devices.

JTD Keywords: flexibility, pedot:pss-alginate hydrogels, self-healing, sustainability, Electrochemical supercapacitors, Flexibility, Pedot:pss-alginate hydrogels, Self-healing, Sustainability

The exponential increase of waste derived from different human activities points out the importance of their reuse in order to create materials with specific properties that can be used for different applications. In this work, it was showed how the typical Mediterranean organic liquid waste, namely olive mill wastewater (OMWW), obtained during olive oil production, can be turned into an efficient reactive agent for the production of noble metals gold (Au) and silver nanoparticles (Ag NPs) with very well-defined physico-chemical properties. More than that, it was demonstrated that this synthetic procedure also leads to a drastic decrease of the organic pollution load of the OMWW, making it safer for environmental disposal and plants irrigation. Then, using healthy hepatic cell line mitochondria, the biological effects induced by these green metal NPs surrounded by a polyphenols shell, with the same NPs synthetized through a standard chemical colloidal reduction process, were compared, finding out that the green NPs are much safer.

JTD Keywords: antioxidants perturbation, green synthesis, gtpase dynamin-related protein 1 expression, mitochondria assessment, physico-chemical properties, Antioxidants perturbation, Green synthesis, Gtpase dynamin-related protein 1 expression, Hepatocytes, Humans, Industrial waste, Metal nanoparticles, Mitochondria, Mitochondria assessment, Olea, Olive oil, Physico-chemical properties, Reusability of waste, Silver, Waste disposal, fluid, Waste water, Wastewater

© 2020, Springer Nature Limited. The human gut microbiome has emerged as a major player in human health and disease. The liver, as the first organ to encounter microbial products that cross the gut epithelial barrier, is affected by the gut microbiome in many ways. Thus, the gut microbiome might play a major part in the development of liver diseases. The common end stage of liver disease is decompensated cirrhosis and the further development towards acute-on-chronic liver failure (ACLF). These conditions have high short-term mortality. There is evidence that translocation of components of the gut microbiota, facilitated by different pathogenic mechanisms such as increased gut epithelial permeability and portal hypertension, is an important driver of decompensation by induction of systemic inflammation, and thereby also ACLF. Elucidating the role of the gut microbiome in the aetiology of decompensated cirrhosis and ACLF deserves further investigation and improvement; and might be the basis for development of diagnostic and therapeutic strategies. In this Review, we focus on the possible pathogenic, diagnostic and therapeutic role of the gut microbiome in decompensation of cirrhosis and progression to ACLF.

JTD Keywords: albumin, decreases intestinal permeability, hepatic-encephalopathy, portal-vein thrombosis, rifaximin improves, secondary bile-acids, systemic inflammation, translocation, venous-pressure gradient, Spontaneous bacterial peritonitis

© 2020 American Chemical Society. The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

JTD Keywords: cancer-on-a-chip, complex, delivery, endothelial-cells, in-vitro, microfluidic, model, nanoparticle, penetration, shear-stress, stability, supramolecular, Cancer-on-a-chip, Cell-culture, Micelle, Microfluidic, Nanoparticle, Stability, Supramolecular

CCBY Intermittent hypoxia (IH) produces autonomic dysfunction that promotes the development of arrhythmia and hypertension in patients with obstructive sleep apnea (OSA). This paper investigated different heart rate variability (HRV) indices in the context of IH using a rat model for OSA. Linear and non-linear HRV parameters were assessed from ultra-short (15-s segments) and short-term (5 min) analyses of heartbeat time-series. Transient changes observed from pre-apnea segments to hypoxia episodes were evaluated, besides the relative and global impact of IH, as a function of its severity. Results showed an overall increase in ultra-short HRV markers as immediate response to hypoxia: standard deviation of normal RR intervals, SDNN=1.2 ms (IQR: 1.1-2.1) vs 1.4 ms (IQR: 1.2-2.2), p=0.015; root mean square of the successive differences, RMSSD=1.7 ms (IQR: 1.5-2.2) vs 1.9 ms (IQR: 1.6-2.4), p=0.031. The power in the very low frequency (VLF) band also showed a significant increase: 0.09 ms2 (IQR: 0.05-0.20) vs 0.16 ms2 (IQR: 0.12-0.23), p=0.016, probably associated with the potentiation of the carotid body chemo-sensory response to hypoxia. Moreover, a clear link between severity of IH and short-term HRV measures was found in VLF and LF power, besides their progressive increase seen throughout the experiment after each apnea sequence. However, only those markers quantifying fragmentation levels in RR series were significantly affected when the experiment ended, as compared to baseline measures: percentage of inflection points, PIP=49% (IQR: 45-51) vs 53% (IQR: 47-53), p=0.031; percentage of short (≥3 RR intervals) accelerated/decelerated segments, PSS=75% (IQR: 51-81) vs 87% (IQR: 51-90), p=0.046. These findings suggest a significant deterioration of cardiac rhythm with a more erratic behavior beyond the normal sinus arrhythmia, that may lead to a future cardiac condition.

JTD Keywords: artificial intelligence, atmospheric modeling, electrocardiography, heart rate variability, hypoxia rat model, intermittent hypoxia, obstructive apneas, protocols, radio access technologies, Artificial intelligence, Atmospheric modeling, Electrocardiography, Heart rate variability, Hypoxia rat model, Intermittent hypoxia, Obstructive apneas, Protocols, Radio access technologies, Rats

In this work, we evaluated a non-linear approach to estimate morphological variations in ECG depolarization, in the context of intermittent hypoxia (IH). Obstructive apnea sequences were provoked for 15 minutes in anesthetized Sprague-Dawley rats, alternating with equal periods of normal breathing, in a recurrent obstructive sleep apnea (OSA) model. Each apnea episode lasted 15 s, while the frequency used for each sequence was randomly selected. Average heartbeats obtained before the start and at the end of each episode, were delineated to extract only the QRS wave. Then, the segmented QRS waves were non-linearly aligned using the dynamic time warping (DWT) algorithm. Morphological QRS changes in both the amplitude and temporal domains were estimated from this alignment procedure. The hypoxic and basal segments were analyzed using ECG (lead I) recordings acquired during the experiment. To assess the effects of IH over time, the changes relative to the basal QRS wave were determined, in the intervals prior to each successive events until the end of the experiment. The results showed a progressive increase in the amplitude and time-domain morphological markers of the QRS wave along the experiment, which were strongly correlated with the changes in traditional QRS markers (r ≈ 0.9). Significant changes were found between pre-apnea and hypoxic measures only for the time-domain analysis (p<0.001), probably due to the short duration of the simulated apnea episodes.Clinical relevance Increased variability in ECG depolarization morphology during recurrent hypoxic episodes would be closely related to the expression of cardiovascular dysfunction in OSA patients.

JTD Keywords: Electrocardiography, Rats, Heart rate variability, Sleep apnea, Protocols, Heuristic algorithms

Chronic obstructive pulmonary disease (COPD) patients exhibit impaired autonomic control which can be assessed by heart rate variability analysis. The study aims to evaluate the cardiac autonomic responses of COPD patients after completing a conventional six-minute walk test (6MWT). Fifty COPD patients were included in the study, for which an ECG signal (lead II) was acquired by a wearable device, before, during, and after the test. We used the heart rate (HR) time-series to assess the heart rate dynamic during recovery. The heart rate recovery (HRR) marker was evaluated every 5 s after the 6MWT and showed different dynamic trends among severity groups. We compared the HRR among patient groups classified according to the GOLD standard. Significantly larger normalized HRR values (nHRR) were found in mild COPD patients (n=23, GOLD={1,2}; nHRR 1 =14.B±7.5 %, nHRR 2 =18.6±8.1 %) compared to those with more disease severity (n=23, GOLD={3,4}; nHRR 1 =9.3±5.8 %, p=0.002; and nHRR 2 = 13.7±6.7%, p=0.041). The largest differences were observed around the first 30 s of the recovery phase (nHRR=10.8±6.6 % vs. nHRR=5.6±4 % p=0.001). Our results showed a slower recovery for the severest patients, suggesting that cardiac parameters like the ones we propose here, may provide valuable information for a better characterization of COPD severity.

JTD Keywords: Pulmonary diseases, Wearable computers, Electrocardiography, Market research, Cardiology, Heart rate variability

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

JTD Keywords: dSTORM, Gene delivery, Polyplexes, Stability, Super-resolution microscopy

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

Cardiovascular diseases are one of the most common causes of death; however, the early detection of patients at high risk of sudden cardiac death (SCD) remains an issue. The aim of this study was to analyze the cardio-vascular couplings based on heart rate variability (HRV) and blood pressure variability (BPV) analyses in order to introduce new indices for noninvasive risk stratification in idiopathic dilated cardiomyopathy patients (IDC). High-resolution electrocardiogram (ECG) and continuous noninvasive blood pressure (BP) signals were recorded in 91 IDC patients and 49 healthy subjects (CON). The patients were stratified by their SCD risk as high risk (IDCHR) when after two years the subject either died or suffered life-threatening complications, and as low risk (IDCLR) when the subject remained stable during this period. Values were extracted from ECG and BP signals, the beat-to-beat interval, and systolic and diastolic blood pressure, and analyzed using the segmented Poincaré plot analysis (SPPA), the high-resolution joint symbolic dynamics (HRJSD) and the normalized short time partial directed coherence methods. Support vector machine (SVM) models were built to classify these patients according to SCD risk. IDCHR patients presented lowered HRV and increased BPV compared to both IDCLR patients and the control subjects, suggesting a decrease in their vagal activity and a compensation of sympathetic activity. Both, the cardio -systolic and -diastolic coupling strength was stronger in high-risk patients when comparing with low-risk patients. The cardio-systolic coupling analysis revealed that the systolic influence on heart rate gets weaker as the risk increases. The SVM IDCLR vs. IDCHR model achieved 98.9% accuracy with an area under the curve (AUC) of 0.96. The IDC and the CON groups obtained 93.6% and 0.94 accuracy and AUC, respectively. To simulate a circumstance in which the original status of the subject is unknown, a cascade model was built fusing the aforementioned models, and achieved 94.4% accuracy. In conclusion, this study introduced a novel method for SCD risk stratification for IDC patients based on new indices from coupling analysis and non-linear HRV and BPV. We have uncovered some of the complex interactions within the autonomic regulation in this type of patient.

JTD Keywords: Idiopathic dilated cardiomyopathy, Heart rate variability, Blood pressure variability, Coupling analysis, Sudden cardiac death, Risk stratification

Background: Many experimental studies have evaluated Linehan’s biological emotional vulnerability in Borderline Personality Disorder (BPD). However, some inconsistencies were observed in operationalizing and supporting its components. This study aims at clarifying which aspects of Linehan’s model are altered in BPD, considering a multimodal evaluation of processes concerned with emotional responsiveness (self-report, psychophysiology and eye-tracking). Methods: Forty-eight socio-emotional pictures were administered to 28 participants (14 BPD, 14 Healthy Controls, HCs), gender- and age-matched, by employing two different lengths of stimuli exposure (5 s and 15 s). Results: Our results supported the hypersensitivity hypothesis in terms of faster physiological responses and altered visual processing. Furthermore, hypersensitivity was associated with detailed socio-emotional contents. Hyperreactivity assumption was not experimentally sustained by physiological and self-report data. Ultimately, the slow return to emotional baseline was demonstrated as an impaired emotional modulation. Conclusions: Our data alternatively supported the hypersensitivity and the slow return to emotional baseline hypotheses, postulated by Linehan’s Biosocial model, rather than the hyperreactivity assumption. Results have been discussed in light of other BPD core psychopathological processes.

JTD Keywords: Borderline personality disorder, Emotional vulnerability, Linehan’s model, Hypersensitivity, Slow return to emotional baseline

Hypoxia induced by obstructive sleep apnea (OSA) leads to the deregulation of the autonomic nervous system (ANS), resulting in an abnormally increased sympathetic activity. Since ANS modulation varies throughout the night, notably for each sleep stage, the hypno-gram and heart rate signals of 81 OSA patients were collected during a polysomnography. They were classified as mild-moderate (n=44) or severe (n=37) based on their apnea-hypopnea index (AHI). Spectral heart rate variability (HRV) series were extracted by a time-frequency approach. These series were then averaged for each sleep stage, in order to compare the sympathetic modulation of mild-moderate and severe patients at the following phases: rapid eye movement (REM), S1, S2 and SWS (slow wave sleep). According to normalized power at the low-frequency band (LFnu) values, severe OSA seems to be associated with an increased sympathetic modulation at non-REM sleep. Moreover, a decreased autonomic variability throughout the night may be related to a reduced adaptability of the cardiovascular system, characterizing a more advanced stage of the disease. These results provide further evidence for the role of autonomic alterations induced by hypoxia, suggesting the use of HRV analysis, together with AHI, for the study of OSA severity.

JTD Keywords: Sleep apnea, Heart rate variability, Modulation, Indexes, Standards

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation. Force-dependent changes in nuclear pores control protein access to the nucleus.

JTD Keywords: Atomic force microscopy, Hippo pathway, Mechanosensing, Mechanotransduction, Molecular mechanical stability, Nuclear mechanics, Nuclear pores, Nuclear transport, Rigidity sensing, Transcription regulation

Calcium phosphate cements (CPC) have seen clinical success in many dental and orthopaedic applications in recent years. The properties of CPC essential for clinical success are reviewed in this article, which includes properties of the set cement (e.g. bioresorbability, biocompatibility, porosity and mechanical properties) and unset cement (e.g. setting time, cohesion, flow properties and ease of delivery to the surgical site). Emphasis is on the delivery of calcium phosphate (CaP) pastes and CPC, in particular the occurrence of separation of the liquid and solid components of the pastes and cements during injection; and established methods to reduce this phase separation. In addition a review of phase separation mechanisms observed during the extrusion of other biphasic paste systems and the theoretical models used to describe these mechanisms are discussed. Statement of Significance Occurrence of phase separation of calcium phosphate pastes and cements during injection limits their full exploitation as a bone substitute in minimally invasive surgical applications. Due to lack of theoretical understanding of the phase separation mechanism(s), optimisation of an injectable CPC that satisfies clinical requirements has proven difficult. However, phase separation of pastes during delivery has been the focus across several research fields. Therefore in addition to a review of methods to reduce phase separation of CPC and the associated constraints, a review of phase separation mechanisms observed during extrusion of other pastes and the theoretical models used to describe these mechanisms is presented. It is anticipated this review will benefit future attempts to develop injectable calcium phosphate based systems.

JTD Keywords: Bone cements, Calcium phosphates, Injectability, Material properties, Phase separation

Although calcium phosphate cements (CPCs) are used for bone regeneration in a wide range of clinical applications, various physicochemical phenomena are known to hinder their potential use in minimally invasive surgery or in highly vascularized surgical sites, mainly because of their lack of injectability or their low washout resistance. The present work shows that the combination of CPCs with an inverse-thermoresponsive hydrogel is a good strategy for finely tuning the cohesive and rheological properties of CPCs to achieve clinical acceptable injectability to prevent phase separation during implantation and cohesion to avoid washout of the paste. The thermoresponsive CPC developed combines alpha-tricalcium phosphate with an aqueous solution of pluronic F127, which exhibits an inverse thermoresponsive behaviour, with a gelling transformation at around body temperature. These novel CPCs exhibited temperature-dependent properties. Addition of the polymer enhanced the injectability of the paste, even at a low liquid-to-powder ratio, and allowed the rheological properties of the cement to be tuned, with the injection force decreasing with the temperature of the paste. Moreover, the cohesion of the paste was also temperature-dependent and increased as the temperature of the host medium increased due to gelling induced in the paste. The thermoresponsive cement exhibited excellent cohesion and clinically acceptable setting times at 37 °C, irrespective of the initial temperature of the paste. The addition of pluronic F127 slightly delayed the setting reaction in the early stages but did not hinder the full transformation to calcium-deficient hydroxyapatite. Moreover, the frozen storage of premixed thermoresponsive cement pastes was explored, the main physicochemical properties of the cements being maintained upon thawing, even after 18 months of frozen storage. This avoids the need to mix the cement in the operating theatre and allows its use off-the-shelf. The reverse thermoresponsive cements studied herein open up new perspectives in the surgical field, where the sequential gelling/hardening of these novel cements could allow for a better and safer clinical application. Statement of Significance: Calcium phosphate cements are attractive bone substitutes due to their similarity to the bone mineral phase. Although they can be injectable, cohesion and stability of the paste are crucial in terms of performance and safety. A common strategy is the combination with hydrogels. However, this often results in a decrease of viscosity with increasing temperature, which can lead to extravasation and particle leakage from the bone defect. The preferred evolution would be the opposite: a low viscosity would enhance mixing and injection, and an instantaneous increase of viscosity after injection would ensure washout resistance to the blood flow. Here we develop for the first time a calcium phosphate cement exhibiting reverse thermoresponsive properties using a poloxamer featuring inverse thermal gelling.

JTD Keywords: Calcium phosphate cement, Cohesion, Hydroxyapatite, Injectability, Pluronic, Thermoresponsive

Graft polymer surfactants provide very good colloidal stability because of strong steric repulsions between adsorbed surfactant films. The elastic and adhesion properties of adsorbed hydrophobically modified inulin polymer surfactant (INUTEC NRA) have been directly measured using Atomic Force Microscopy (AFM) measurements. For this purpose, poly(methyl methacrylate/butyl acrylate), P(MMA/BuA), latexes prepared in the presence of the hydrophobically modified inulin (INUTEC NRA) were used. These latexes (diameter 118 nm and polydispersity index of 1.05) showed a very high colloidal stability in water and in the presence of electrolyte (up to 0.2 mol dm−3 KBr). The latexes were deposited on mica, which was silanated to enhance the adhesion of the latex particles to the surface. A silicon nitride tip with approximately 10 nm diameter that also contained an adsorbed layer of surfactant was used in the AFM apparatus. The tip was allowed to approach, contact thereafter the particles with an applied force of 12.5 nN, and finally detach from the film. Both elastic (Young’s) modulus of the film and adhesion force were studied. The results showed that the adsorbed surfactant films are highly elastic and their elastic modulus and adhesion force did not change significantly with the presence of Na2SO4 up to 0.05 mol dm−3. The high elastic contribution to the steric interaction ensures strong repulsion between the latex particles both in water and at high electrolyte concentrations. In addition, the lack of dependence of adhesion force on electrolyte concentration ensures uniform deposition of the latex particles on a flat substrate as for example in coating applications. These results show the advantages of using a graft polymer surfactant for enhancing the stability of particle suspensions, as illustrated in previous investigations.

JTD Keywords: AFM, Colloidal stability, Interaction forces, Steric repulsion

Global regulators play an essential role in the adaptation of bacterial cells to specific niches. Bacterial pathogens thriving in the tissues and organs of their eukaryotic hosts are a well-studied example. Some of the proteins that recognize local DNA structures rather than specific nucleotide sequences act as global modulators in many bacteria, both Gram-negative and -positive. To this class of regulators belong the H-NS-like proteins, mainly identified in γ-Proteobacteria, and the MgaSpn-like proteins identified in Firmicutes. H-NS and MgaSpn from Escherichia coli and Streptococcus pneumoniae, respectively, neither have sequence similarity nor share structural domains. Nevertheless, they display common features in their interaction with DNA, namely: (i) they bind to DNA in a non-sequence-specific manner, (ii) they have a preference for intrinsically curved DNA regions, and (iii) they are able to form multimeric complexes on linear DNA. Using DNA fragments from the hemolysin operon regulatory region of the E. coli plasmid pHly152, we show in this work that MgaSpn is able to recognize particular regions on extended H-NS binding sites. Such regions are either located at or flanked by regions of potential bendability. Moreover, we show that the regulatory region of the pneumococcal P1623B promoter, which is recognized by MgaSpn, contains DNA motifs that are recognized by H-NS. These motifs are adjacent to regions of potential bendability. Our results suggest that both regulatory proteins recognize similar structural characteristics of DNA.

JTD Keywords: Global transcriptional regulators, Nucleoid-associated proteins, Mga/AtxA family, Protein-DNA interactions, DNA bendability

Aim: Explore the use of polymeric micelles in the development of powders intended for pulmonary delivery of biopharmaceuticals, using insulin as a model protein. Materials & methods: Formulations were assessed in vitro for aerosolization properties and in vivo for efficacy and safety using a streptozotocin-induced diabetic rat model. Results: Powders presented good aerosolization properties like fine particle fraction superior to 40% and a mass median aerodynamic diameter inferior of 6 μm. Endotracheally instilled powders have shown a faster onset of action than subcutaneous administration of insulin at a dose of 10 IU/kg, with pharmacological availabilities up to 32.5% of those achieved by subcutaneous route. Additionally, micelles improved the hypoglycemic effect of insulin. Bronchoalveolar lavage screening for toxicity markers (e.g., lactate dehydrogenase, cytokines) revealed no signs of lung inflammation and cytotoxicity 14 days postadministration. Conclusion: Developed powders showed promising safety and efficacy characteristics for the systemic delivery of insulin by pulmonary administration.

JTD Keywords: Fine particle fraction, Inhalation, Insulin, In vivo, Pharmacological availability, Polymeric micelles, Pulmonary toxicity

IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.

JTD Keywords: Antimicrobial resistance, E protein, IncC, IncHI1 plasmids, Plasmid R27, Plasmid stability

Pulmonary delivery of drugs for both local and systemic action has gained new attention over the last decades. In this work, different amphiphilic polymers (Soluplus®, Pluronic® F68, Pluronic® F108 and Pluronic® F127) were used to produce lyophilized formulations for inhalation of insulin. Development of stimuli-responsive, namely glucose-sensitive, formulations was also attempted with the addition of phenylboronic acid (PBA). Despite influencing the in vitro release of insulin from micelles, PBA did not confer glucose-sensitive properties to formulations. Lyophilized powders with aerodynamic diameter (<. 6. μm) compatible with good deposition in the lungs did not present significant in vitro toxicity for respiratory cell lines. Additionally, some formulations, in particular Pluronic® F127-based formulations, enhanced the permeation of insulin through pulmonary epithelial models and underwent minimal internalization by macrophages in vitro. Overall, formulations based on polymeric micelles presenting promising characteristics were developed for the delivery of insulin by inhalation. From the Clinical Editor: The ability to deliver other systemic drugs via inhalation has received renewed interests in the clinical setting. This is especially true for drugs which usually require injections for delivery, like insulin. In this article, the authors investigated their previously developed amphiphilic polymers for inhalation of insulin in an in vitro model. The results should provide basis for future in vivo studies.

JTD Keywords: Cytotoxicity, Inhalation, Permeability, Phagocytosis, Polymeric micelles, Protein delivery

Abstract Nanocomposite powders composed by polymeric micelles as vehicles for delivery proteins were developed in this work, using insulin as model protein. Results showed that size and polydispersity of micelles were dependent on the amphiphilic polymer used, being all lower than 300 nm, while all the formulations displayed spherical shape and surface charge close to neutrality. Percentages of association efficiency and loading capacity up to 94.15 ± 3.92 and 8.56 ± 0.36, respectively, were obtained. X-ray photoelectron spectroscopy (XPS) measurements confirmed that insulin was partially present at the hydrophilic shell of the micelles. Lyophilization did not significantly change the physical characteristics of micelles, further providing easily dispersion when in contact to aqueous medium. The native-like conformation of insulin was maintained at high percentages (around 80%) after lyophilization as indicated by Fourier transform infrared spectroscopy (FTIR) and far-UV circular dichroism (CD). Moreover, Raman spectroscopy did not evidenced significant interactions among the formulation components. The formulations shown to be physically stable upon storage up to 6 months both at room-temperature (20 C) and fridge (4 C), with only a slight loss (maximum of 15%) of the secondary structure of the protein. Among the polymers tested, Pluronic® F127 produced the carrier formulations more promising for delivery of proteins.

JTD Keywords: Amphiphilic polymers, Insulin, Lyophilization, Polymeric micelles, Stability

Abstract Wearable robots are expected to expand the use of robotics in rehabilitation since they can widen the assistance application context. An important aspect of a rehabilitation therapy, in terms of lower extremity assistance, is balance control. In this article, we propose and evaluate an adaptive control strategy for robotic rehabilitation therapies to guarantee static stability using a wearable robot. Postural balance control can be implemented either acting on the hip, on the ankle joint or on both, depending on the kind of perturbation acting on the subject: internal or external. Internal perturbations can be produced by any voluntary movement of the body, such as bending the trunk. External perturbations, in the form of an impact force, are applied by the exoskeleton without any prior notice to observe the proactive response of the subject. We have used a 6 degree of freedom planar lower limb exoskeleton, H1, to perform this analysis. The developed control strategy has been designed to provide the necessary assistance, related to balance recovery and postural stability, under the “Assist-as-needed” paradigm. The interaction forces between orthosis and subject are monitored, as they play a relevant role in the definition of assistive and resistive movements to be applied to the joints. The proposed method has been tested with 5 healthy subjects in presence of internal and external disturbances. The results demonstrate that knowing the stability limit of each subject, in combination with a therapeutically selected scaling factor, the proposed adaptive control helps in providing an effective assistance in therapy. This method is efficient in handling the individual and combined effect of external perturbations acting on any joint movements.

JTD Keywords: Exoskeleton controls, Postural stability, Balance controls, Adaptive control

Electric Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present a study on the effects of electric field intensity and temperature to bacterial cultures. Escherichia coli colonies have been exposed to different electric field intensities at 1MHz during 5 minutes by means of a microfluidic device specially designed for the experiment. From the analysis of the results it is possible to see that Escherichia coli survival rate diminishes when applying field intensities as low as 220V during 5 minutes. Death rates also increase when stronger fields are applied. However, viability of survived bacteria is maintained. Additionally, temperature shows a synergistic effect with voltage. When temperature was increased, results showed a stronger sensitivity of cells to the electric field. Moreover, the expression patterns of Outer Membrane Protein A and Ribosomal Proteins differ in control and treated samples, suggesting changes in bacterial metabolism and structure.

JTD Keywords: E. coli, Electric field, Temperature, Viability

Objective To investigate the relevance of the human vertebral endplate poromechanics on the fluid and metabolic transport from and to the intervertebral disc (IVD) based on educated estimations of the poromechanical parameter values of the bony endplate (BEP). Methods 50 micro-models of different BEP samples were generated from μCTs of lumbar vertebrae and allowed direct determination of porosity values. Permeability values were calculated by using the micro-models, through the simulation of permeation via computational fluid dynamics. These educated ranges of porosity and permeability values were used as inputs for mechano-transport simulations to assess their effect on both the distributions of metabolites within an IVD model and the poromechanical calculations within the cartilaginous part of the endplate i.e., the cartilage endplate (CEP). Results BEP effective permeability was highly correlated to local variations of porosity (R2 ≈ 0.88). Universal patterns between bone volume fraction and permeability arose from these results and from other experimental data in the literature. These variations in BEP permeability and porosity had negligible effects on the distributions of metabolites within the disc. In the CEP, the variability of the poromechanical properties of the BEP did not affect the predicted consolidation but induced higher fluid velocities. Conclusions The present paper provides the first sets of thoroughly identified BEP parameter values that can be further used in patient-specific poromechanical studies. Representing BEP structural changes through variations in poromechanical properties did not affect the diffusion of metabolites. However, attention might be paid to alterations in fluid velocities and cell mechano-sensing within the CEP.

JTD Keywords: Bony endplate, Spine mechanobiology, Intervertebral disc metabolites, Hydraulic Permeability, Bone Porosity, Poromechanics

Novel strategies to heal discogenic low back pain could highly benefit from comprehensive biophysical studies that consider both mechanical and biological factors involved in intervertebral disc degeneration. A decrease in nutrient availability at the bone-disc interface has been indicated as a relevant risk factor and as a possible initiator of cell death processes. Mechanical behaviour of both healthy and degenerated discs could highly interact with cell death in these compromised situations. In the present study, a mechano-transport finite element model was used to investigate the nature of mechanical effects on cell death processes via load-induced metabolic transport variations. Cycles of static sustained compression were chosen to simulate daily human activity. Healthy and degenerated cases were simulated as well as a reduced supply of solutes and an increase in solute exchange area at the bone-disc interface. Results showed that a reduction in metabolite concentrations at the bone-disc boundaries induced cell death, even when the increased exchange area was simulated. Slight local mechanical enhancements of glucose in the disc centre were capable of decelerating cell death but occurred only with healthy mechanical properties. However, mechanical deformations were responsible for a worsening in terms of cell death in the inner annulus, a disadvantaged zone far from the boundary supply with both an increased cell demand and a strain-dependent decrease of diffusivity. Such adverse mechanical effects were more accentuated when degenerative properties were simulated. Overall, this study paves the way for the use of biophysical models for a more integrated understanding of intervertebral disc pathophysiology.

JTD Keywords: Boundary conditions, Cell nutrition, Cell viability, Computational analysis, Intervertebraldisc, Softtissuebiomechanics

Poly(vinylchloride) (PVC) is the most common polymer matrix used in the fabrication of ion-selective electrodes (ISEs). However, the surfaces of PVC-based sensors have been reported to show membrane instability. In an attempt to overcome this limitation, here we developed two alternative methods for the preparation of highly stable and robust ion-selective sensors. These platforms are based on the selective electropolymerization of poly(3,4-ethylenedioxythiophene) (PEDOT), where the sulfur atoms contained in the polymer covalently interact with the gold electrode, also permitting controlled selective attachment on a miniaturized electrode in an array format. This platform sensor was improved with the crosslinking of the membrane compounds with poly(ethyleneglycol) diglycidyl ether (PEG), thus also increasing the biocompatibility of the sensor. The resulting ISE membranes showed faster signal stabilization of the sensor response compared with that of the PVC matrix and also better reproducibility and stability, thus making these platforms highly suitable candidates for the manufacture of robust implantable sensors.

JTD Keywords: Biomedicine, Electrochemistry, Endoscope, Implantable device, Ion-selective electrode (ISE) sensor, Ischemia, pH detection, Biocompatibility, Chemical sensors, Electrochemistry, Electrodes, Electropolymerization, Endoscopy, Functional polymers, Implants (surgical), Ion selective electrodes, Medical applications, Polyvinyl chlorides, Stabilization, Biomedical applications, Biomedicine, Implantable devices, Ion selective sensors, Ischemia, Membrane instability, pH detection, Poly(3 ,4 ethylenedioxythiophene) (PEDOT), Ion selective membranes

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro-and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules-DNA or proteins-to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.

JTD Keywords: Atomic force microscopy, Cations, Force spectroscopy, Lipid bilayer, Mechanical stability

Blood pressure carries a great deal of information about people’s physical attributes. We analyzed the blood pressure signal in healthy subjects considering two positions, supine and sitting. 44 healthy subjects were studied. Parameters extracted from the blood pressure signal, related to time and frequency domain were used to compare the effect of postural position between supine and sitting. In time domain analysis, the time systolic interval and the time of blood pressure interval were higher in supine than in sitting position (p = 0.001 in both case). Parameters related to frequency peak, interquartile range, in frequency domain presented statistically significant difference (p < 0.0005 in both case). The blood pressure variability parameters presented smaller values in supine than in sitting position (p < 0.0005). In general, the position change of supine to sitting produces an increment in the pressure gradient inside heart, reflected in the blood pressure variability.

JTD Keywords: Blood pressure variability, Systolic time intervals, Diastolic time intervals

One of the challenges of the robotics community is to develop robots that behave more and more autonomously. Therefore, it is necessary to establish new design criteria, as well as more complex methodologies supporting the analysis of associated risks. The procedure described in this paper includes civil liability as an additional criterion to validate the safety of a surgical robot. In order to understand the concept, a methodology is presented through the description of a simple case. This work aims to establish the basis for a further implementation.

JTD Keywords: Design methodology, Product development, Product liability, Safety, Robotic surgery, Cognitive robotics

Electromagnetic Fields are increasingly used to manipulate bacteria. However, there is no systematic and definitive study on how the different electric parameters change bacteria viability. Here we present preliminary data on the effect of electric field intensity and temperature applica- tion. E. Coli colonies have been exposed to different voltages at 1MHz during 5 minutes by means of a custom-made micro- fluidic device. Results show that E.Coli survival rate is already reduced by applying field intensities as low as 220V/cm during 5 minutes. The use of stronger fields resulted in death rates increase also. Viability of survived bacteria was maintained. On the other hand, temperature has shown a synergistic effect with voltage. When temperature is increased results seem to indicate stronger sensitivity of cells to the electric field. It is necessary to continue studying the contribution of other para- meters as intensity, time, frequency or concentration, to study further synergies.

JTD Keywords: E. Coli, Electromagnetic Field, Temperature, Viability

The aim of the present work was to analyze the influence of the setting reaction on the injectability of tricalcium phosphate (TCP) pastes. Even if the injection was performed early after mixing powder and liquid, powder reactivity was shown to play a significant role in the injectability of TCP pastes. Significant differences were observed between the injection behavior of non-hardening β-TCP pastes and that of self-hardening α-TCP pastes. The differences were more marked at low liquid-to-powder ratios, using fine powders and injecting through thin needles. α-TCP was, in general, less injectable than β-TCP and required higher injection loads. Moreover, clogging was identified as a mechanism hindering or even preventing injectability, different and clearly distinguishable from the filter-pressing phenomenon. α-TCP pastes presented transient clogging episodes, which were not observed in β-TCP pastes with equivalent particle size distribution. Different parameters affecting powder reactivity were also shown to affect paste injectability. Thus, whereas powder calcination resulted in an increased injectability due to lower particle reactivity, the addition of setting accelerants, such as hydroxyapatite nanoparticles, tended to reduce the injectability of the TCP pastes, especially if adjoined simultaneously with a Na2HPO4 solution. Although, as a general trend, faster-setting pastes were less injectable, some exceptions to this rule were found. For example, whereas in the absence of setting accelerants fine TCP powders were more injectable than the coarse ones, in spite of their shorter setting times, this trend was inverted when setting accelerants were added, and coarse powders were more injectable than the fine ones.

JTD Keywords: Calcium phosphate cement, Hydroxyapatite, Injectability, Setting reaction, Tricalcium phosphate

STUDY QUESTION Is the attachment of biofunctionalized polysilicon barcodes to the outer surface of the zona pellucida an effective approach for the direct tagging and identification of cultured embryos? SUMMARY ANSWER The results achieved provide a proof of concept for a direct embryo tagging system using biofunctionalized polysilicon barcodes, which could help to minimize the risk of mismatching errors (mix-ups) in human assisted reproduction technologies. WHAT IS KNOWN ALREADY Even though the occurrence of mix-ups is rare, several cases have been reported in fertility clinics around the world. Measures to prevent the risk of mix-ups in human assisted reproduction technologies are therefore required. STUDY DESIGN, SIZE, DURATION Mouse embryos were tagged with 10 barcodes and the effectiveness of the tagging system was tested during fresh in vitro culture (n=140) and after embryo cryopreservation (n = 84). Finally, the full-term development of tagged embryos was evaluated (n =105). PARTICIPANTS/ MATERIALS, SETTING, METHODS Mouse pronuclear embryos were individually rolled over wheat germ agglutinin-biofunctionalized polysilicon barcodes to distribute them uniformly around the ZONA PELLUCIDA surface. Embryo viability and retention of barcodes were determined during 96 h of culture. The identification of tagged embryos was performed every 24 h in an inverted microscope and without embryo manipulation to simulate an automatic reading procedure. Full-term development of the tagged embryos was assessed after their transfer to pseudo-pregnant females. To test the validity of the embryo tagging system after a cryopreservation process, tagged embryos were frozen at the 2-cell stage using a slow freezing protocol, and followed in culture for 72 h after thawing. MAIN RESULTS AND THE ROLE OF CHANCE Neither the in vitro or in vivo development of tagged embryos was adversely affected. The tagging system also proved effective during an embryo cryopreservation process. Global identification rates higher than 96 and 92% in fresh and frozen-thawed tagged embryos, respectively, were obtained when simulating an automatic barcode reading system, although these rates could be increased to 100% by simply rotating the embryos during the reading process. LIMITATIONS, REASONS FOR CAUTION The direct embryo tagging developed here has exclusively been tested in mouse embryos. Its effectiveness in other species, such as the human, is currently being tested. WIDER IMPLICATIONS OF THE FINDINGS The direct embryo tagging system developed here, once tested in human embryos, could provide fertility clinics with a novel tool to reduce the risk of mix-ups in human assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Spanish Ministry of Education and Science (TEC2011-29140-C03) and by the Generalitat de Catalunya (2009SGR-00282).

JTD Keywords: Assisted reproductive technologies (ART), Biofunctionalization, Embryo tagging, Mix-ups, Traceability

Assessment of the dynamic interactions between cardiovascular signals can provide valuable information that improves the understanding of cardiovascular control. Heart rate variability (HRV) analysis is known to provide information about the autonomic heart rate modulation mechanism. Using the HRV signal, we aimed to obtain parameters for classifying patients with and without chronic heart failure (CHF), and with periodic breathing (PB), non-periodic breathing (nPB), and Cheyne-Stokes respiration (CSR) patterns. An electrocardiogram (ECG) and a respiratory flow signal were recorded in 36 elderly patients: 18 patients with CHF and 18 patients without CHF. According to the clinical criteria, the patients were classified into the follow groups: 19 patients with nPB pattern, 7 with PB pattern, 4 with Cheyne-Stokes respiration (CSR), and 6 non-classified patients (problems with respiratory signal). From the HRV signal, parameters in the time and frequency domain were calculated. Frequency domain parameters were the most discriminant in comparisons of patients with and without CHF: P_Tot (p = 0.02), P_LF (p = 0.022) and fp_HF (p = 0.021). For the comparison of the nPB vs. CSR patients groups, the best parameters were RMSSD (p = 0.028) and SDSD (p = 0.028). Therefore, the parameters appear to be suitable for enhanced diagnosis of decompensated CHF patients and the possibility of developed periodic breathing and a CSR pattern.

JTD Keywords: cardiovascular system, diseases, electrocardiography, frequency-domain analysis, geriatrics, medical signal processing, patient diagnosis, pneumodynamics, signal classification, Cheyne-Stokes respiration patterns, ECG, autonomic heart rate modulation mechanism, cardiovascular control, cardiovascular signals, chronic heart failure, decompensated CHF patients, dynamic interaction assessment, elderly patients, electrocardiogram, enhanced diagnosis, frequency domain parameters, heart rate variability analysis, patient classification, periodic breathing, respiratory flow signal recording, Electrocardiography, Frequency modulation, Frequency-domain analysis, Heart rate variability, Senior citizens, Standards

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

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

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

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

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

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

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

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

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational- active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca 2+ resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

JTD Keywords: Cumulative probability distribution, Integrin lymphocyte function-associated antigen 1, Intercellular adhesion molecule, Single molecule detection

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si 3N 4) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si 3N 4-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO 2/Si 3N 4 structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10 -13-10 -7M were detected, showing a sensitivity of 0.128ΩμM -1 and a limit of detection of 10 -14M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.

JTD Keywords: Aldehyde, Electrochemical impedance spectroscopy, Human serum albumin, Immunosensor, Silicon nitride, Bovine serum albumins, Chemical reagents, Complex samples, Covalent binding, Detection capability, Electrochemical impedance, Electrochemical impedance spectroscopy measurements, Functionalizations, Human serum albumins, Impedimetric immunosensors, Label free, Limit of detection, Linear range, Protein concentrations, Silicon-based, Specific detection, Aldehydes

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.

JTD Keywords: Chain-Length, Spectroscopy, Nanomechanics, Microscopy, Elasticity, Stability, Membranes, Reveals, Fusion, Ions

Proteins are fundamental molecules in biology that are also involved in a wide range of industrial and biotechnological processes. Consequently, many works in the literature have been devoted to the study of protein-protein and protein-surface interactions in aqueous solutions. The results have been usually interpreted within the frame of the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for colloidal systems. However, against the DLVO predictions, striking evidence of repulsive forces between proteins at high salt concentrations has been observed in different works based on the analysis of the second virial coefficient or on the direct measurement of protein interaction with an atomic force microscope. Hydration forces due to the adsorption of hydrated cations onto the negatively charged protein surfaces have been invoked to rationalize this anomalous repulsion. The hydration forces between proteins provide protein-covered particles with a non-DLVO colloidal stability at high salt concentrations, as different studies in the literature has proven. This review summarizes the most relevant results published so far on the presence of hydration forces between proteins and protein-coated colloidal particles.

JTD Keywords: Colloidal particles, Colloidal stability, Hydrated ions, Hydration forces, Proteins

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition.

JTD Keywords: Bovine nucleus pulposus, Human anulus fibrosus, Finite-element, Fluid-flow, Hydraulic permeability, Confined compression, Coupled diffusion, Solute transport, Water-content, Lumbar spine

Despite their known osteoconductivity, clinical use of calcium phosphate cements is limited both by their relatively slow rate of resorption and by rheological properties incompatible with injectability. Bone in-growth and material resorption have been improved by the development of porous calcium phosphate cements. However, injectable formulations have so far only been obtained through the addition of relatively toxic surfactants. The present work describes the response of osteoblasts to a novel injectable foamed bone cement based on a composite formulation including the bioactive foaming agents soybean and gelatine. The foaming properties of both defatted soybean and gelatine gels were exploited to develop a self-hardening soy/gelatine/hydroxyapatite composite foam able to retain porosity upon injection. After setting, the foamed paste produced a calcium-deficient hydroxyapatite scaffold, showing good injectability and cohesion as well as interconnected porosity after injection. The intrinsic bioactivity of soybean and gelatine was shown to favour osteoblast adhesion and growth. These findings suggest that injectable, porous and bioactive calcium phosphate cements can be produced for bone regeneration through minimally invasive surgery.

JTD Keywords: Calcium phosphate cement, Composite, Bone tissue engineering, Cell viability, Bioactivity

Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH) = 0, X(OH) = 0.3, X(OH) = 0.5, X(OH) = 0.7, and X(OH) = 1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH) = 1 and other on the relatively hydrophobic X(OH) = 0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH) = 0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.

JTD Keywords: Atomic-force microscopy, Fibronectin adsorption, Basement-membranes, Polymer surfaces, Cell-adhesion, Biomaterials, Wettability, Fibrinogen

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

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

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

JTD Keywords: Sensors, Inference mechanisms, Probability, Simulation

This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PM MA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToRSIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SA V) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.

JTD Keywords: Wettability gradient, Polyethylene surface, Combinatorial, Immobilization, Biomaterials, Fabrication, Deposition, Bradykinin, Monolayers, Discharge

An important goal in cluster analysis is the internal validation of results using an objective criterion. Of particular relevance in this respect is the estimation of the optimum number of clusters capturing the intrinsic structure of your data. This paper proposes a method to determine this optimum number based on the evaluation of fuzzy partition stability under bootstrap resampling. The method is first characterized on synthetic data with respect to hyper-parameters, like the fuzzifier, and spatial clustering parameters, such as feature space dimensionality, clusters degree of overlap, and number of clusters. The method is then validated on experimental datasets. Furthermore, the performance of the proposed method is compared to that obtained using a number of traditional fuzzy validity rules based on the cluster compactness-to-separation criteria. The proposed method provides accurate and reliable results, and offers better generalization capabilities than the classical approaches.

JTD Keywords: Fuzzy c-means, Cluster validity, Number of clusters, Cluster stability

A simple method to measure the static dielectric constant of thin films with nanometric spatial resolution is presented. The dielectric constant is extracted from DC electrostatic force measurements with the use of an accurate analytical model. The method is validated here on thin silicon dioxide films (8 nm thick, dielectric constant approximately 4) and purple membrane monolayers (6 nm thick, dielectric constant approximately 2), providing results in excellent agreement with those recently obtained by nanoscale capacitance microscopy using a current-sensing approach. The main advantage of the force detection approach resides in its simplicity and direct application on any commercial atomic force microscope with no need of additional sophisticated electronics, thus being easily available to researchers in materials science, biophysics and semiconductor technology.

JTD Keywords: Roscopy, Membrane, Tip, Polarizability, Polarization, Resolution, Nanotubes, Charge

A statistical factorial analysis approach was conducted on a poroelastic finite element model of a lumbar intervertebral disc to analyse the influence of six material parameters (permeabilities of annulus, nucleus, trabecular vertebral bone, cartilage endplate and Young's moduli of annulus and nucleus) on the displacement, fluid pore pressure and velocity fields. Three different loading modes were investigated: compression, flexion and axial rotation. Parameters were varied considering low and high levels in agreement with values found in the literature for both healthy and degenerated lumbar discs. Results indicated that annulus stiffness and cartilage endplate permeability have a strong effect on the overall fluid- and solid-phase responses in all loading conditions studied. Nucleus stiffness showed its main relevance in compression while annulus permeability influenced mainly the annular pressure field. This study confirms the permeability's central role in biphasic modelling and highlights for the lumbar disc which experiments of material property characterization should be performed. Moreover, such sensitivity study gives important guidelines in poroelastic material modelling and finite element disc validation.

JTD Keywords: Intervertebral disc, Permeability, Fractional factorial design, Design of experiments, Finite element analysis

Here, the layer-by-layer technique (LbL) was used to modify glass as model biomaterial with multilayers of chitosan and heparin to control the interaction with MG-63 osteoblast-like cells. Different pH values during multilayer formation were applied to control their physico-chemical properties. In the absence of adhesive proteins like plasma fibronectin (pFN) both plain layers were rather cytophobic. Hence, the preadsorption of pFN was used to enhance cell adhesion which was strongly dependent on pH. Comparing the adhesion promoting effects of pFN with an engineered repeat of the FN III fragment and collagen I which both lack a heparin binding domain it was found that multilayers could bind pFN specifically because only this protein was capable of promoting cell adhesion. Multilayer surfaces that inhibited MG-63 adhesion did also cause a decreased cell growth in the presence of serum, while an enhanced adhesion of cells was connected to an improved cell growth.

JTD Keywords: Cell-adhesion, Polyelectrolyte multilayers, Substratum chemistry, Surface-properties, Fibroblast-growth, Fibronectin, Polymers, Chitosan, Polysaccharides, Wettability

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds' success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds' micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells' behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.

JTD Keywords: Biocompatible Materials/ chemistry, Bone and Bones/ metabolism, Calcium Phosphates/ chemistry, Cell Differentiation, Cell Proliferation, Humans, Lactic Acid/ chemistry, Microscopy, Confocal, Microscopy, Electron, Scanning, Osteoblasts/metabolism, Permeability, Polymers/ chemistry, Porosity, Solvents/chemistry, Tissue Engineering/ methods

In this study polymer-glass composite scaffolds were characterized by permeability and porosity, two important properties for the use in perfusion bioreactors. These scaffolds were seeded with osteoblast-like cells to assess the efficiency of the used bioreactor. The used PLA/glass composite scaffolds are adequate for the perfusion culture. The high porosity and pore interconnectivity allow an even cell distribution and incorporation of a high cell number. For optimisation of the perfusion bioreactor system, further research has to be dedicated to the cell seeding and culture.

JTD Keywords: Biomedical materials, Bioreactors, Bone, Cellular biophysics, Composite materials, Orthopaedics, Permeability, Polymers, Porosity, Porous materials, Tissue engineering

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

JTD Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

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JTD Keywords: Self-assembled monolayers, High-aspect-ratio, Dip-pen nanolithography, Langmuir-blodgett-films, Conducting polymer, Enzymes, Microstructures, Immunosensors, Fabrication, Stability