Interactions Between Silver Nanoparticles and Culture Medium Biomolecules with Dose and Time Dependencies.

The interaction between silver nanoparticles (AgNPs) and molecules producing coronas plays a key role in cytotoxicity mechanisms. Once adsorbed coronas determine the destiny of nanomaterials in vivo, their effective deployment in the biomedical field requires a comprehensive understanding of the dynamic interactions of biomolecules with nanoparticles. In this work, we characterized 40 nm AgNPs in three different nutritional cell media at different molar concentrations and incubation times to study the binding mechanism of molecules on surface nanoparticles. In addition, their cytotoxic effects have been studied in three cell lineages used as tissue regeneration models: FN1, HUV-EC-C, RAW 264.7. According to the data, when biomolecules from DMEM medium were in contact with AgNPs, agglomeration and precipitation occurred. However, FBS medium proteins indicated the formation of coronas over the nanoparticles. Nonetheless, little adsorption of molecules around the nanoparticles was observed when compared to DMEM supplemented with 10% FBS. These findings indicate that when nanoparticles and bioproteins from supplemented media interact, inorganic salts from DMEM contribute to produce large bio-coronas, the size of which varies with the concentration and time. The static quenching mechanism was shown to be responsible for the fluorescence quenching of the bioprotein aggregates on the AgNPs surface. The calculated bioprotein-nanoparticle surface binding constants were on the order of 105 M-1 at 37 °C, with hydrophobic interactions driven by enthalpy and entropy playing a role, as confirmed by thermodynamic analysis. Cytotoxicity data showed a systematic degrowth in the viable cell population as the number of nanoparticles increased and the diameter of coronas decreased. Cytotoxic intervals associated with half decrease of cell population were established for AgNPs molar concentration of 75 µM for 24 h and 50 µM for 48 h. In summary, through the cytotoxicity mechanism of bio-coronas we are able to manipulate cells' expansion rates to promote specific processes, such inflammatory mechanisms, at different time instants.

Peri/epicellular protein disulfide isomerase-A1 acts as an upstream organizer of cytoskeletal mechanoadaptation in vascular smooth muscle cells.

Although redox processes closely interplay with mechanoresponses to control vascular remodeling, redox pathways coupling mechanostimulation to cellular cytoskeletal organization remain unclear. The peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) supports postinjury vessel remodeling. Using distinct models, we investigated whether pecPDIA1 could work as a redox-dependent organizer of cytoskeletal mechanoresponses. In vascular smooth muscle cells (VSMCs), pecPDIA1 immunoneutralization impaired stress fiber assembly in response to equibiaxial stretch and, under uniaxial stretch, significantly perturbed cell repositioning perpendicularly to stretch orientation. During cyclic stretch, pecPDIA1 supported thiol oxidation of the known mechanosensor ß1-integrin and promoted polarized compartmentalization of sulfenylated proteins. Using traction force microscopy, we showed that pecPDIA1 organizes intracellular force distribution. The net contractile moment ratio of platelet-derived growth factor-exposed to basal VSMCs decreased from 0.90 ± 0.09 (IgG-exposed controls) to 0.70 ± 0.08 after pecPDI neutralization ( P < 0.05), together with an enhanced coefficient of variation for distribution of force modules, suggesting increased noise. Moreover, in a single cell model, pecPDIA1 neutralization impaired migration persistence without affecting total distance or velocity, whereas siRNA-mediated total PDIA1 silencing disabled all such variables of VSMC migration. Neither expression nor total activity of the master mechanotransmitter/regulator RhoA was affected by pecPDIA1 neutralization. However, cyclic stretch-induced focal distribution of membrane-bound RhoA was disrupted by pecPDI inhibition, which promoted a nonpolarized pattern of RhoA/caveolin-3 cluster colocalization. Accordingly, FRET biosensors showed that pecPDIA1 supports localized RhoA activity at cell protrusions versus perinuclear regions. Thus, pecPDI acts as a thiol redox-dependent organizer and noise reducer mechanism of cytoskeletal repositioning, oxidant generation, and localized RhoA activation during a variety of VSMC mechanoresponses. NEW & NOTEWORTHY Effects of a peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) during mechanoregulation in vascular smooth muscle cells (VSMCs) were highlighted using approaches such as equibiaxial and uniaxial stretch, random single cell migration, and traction force microscopy. pecPDIA1 regulates organization of the cytoskeleton and minimizes the noise of cell alignment, migration directionality, and persistence. pecPDIA1 mechanisms involve redox control of ß1-integrin and localized RhoA activation. pecPDIA1 acts as a novel organizer of mechanoadaptation responses in VSMCs.

Integrated molecular, biochemical, and physiological assessment unravels key extraction method mediated influences on rat neonatal cardiomyocytes.

Neonatal cardiomyocytes are instrumental for disease modeling, but the effects of different cell extraction methods on basic cell biological processes remain poorly understood. We assessed the influence of two popular methods to extract rat neonatal cardiomyocytes, Pre-plating (PP), and Percoll (PC) on cell structure, metabolism, and function. Cardiomyocytes obtained from PP showed higher gene expression for troponins, titin, and potassium and sodium channels compared to PC. Also, PP cells displayed higher levels of troponin I protein. Cells obtained from PC displayed higher lactate dehydrogenase activity and lactate production than PP cells, indicating higher anaerobic metabolism after 8 days of culture. In contrast, reactive oxygen species levels were higher in PP cells as indicated by ethidium and hydroxyethidium production. Consistent with these data, protein nitration was higher in PP cells, as well as nitrite accumulation in cell medium. Moreover, PP cells showed higher global intracellular calcium under basal and 1 mM isoprenaline conditions. In a calcium-transient assessment under electrical stimulation (0.5 Hz), PP cells displayed higher calcium amplitude than cardiomyocytes obtained from PC and using a traction force microscope technique we observed that PP cardiomyocytes showed the highest relaxation. Collectively, we demonstrated that extraction methods influence parameters related to cell structure, metabolism, and function. Overall, PP derived cells are more active and mature than PC cells, displaying higher contractile function and generating more reactive oxygen species. On the other hand, PC derived cells display higher anaerobic metabolism, despite comparable high yields from both protocols.

Quantification of alignment of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are essential components that keep the tonus of the arterial network, which is the channel used to conduct the blood from the heart to the peripheral areas of the body. It is known that mechanical and architectural changes in VSMCs may lead to functional modifications in the cardiovascular system; therefore, the quantitative characterization of these changes can help to elucidate questions that remain unclear in pathological situations, such as hypertension, vasospasm, vascular hypertrophy, and atherosclerosis. In this work, we have developed a new framework of image processing using the Sobel operator, associated with statistical analysis, to determine the degree of local alignment of actin filaments, which we found to be directly related with the distensibility of the arterial wall. We have also compared these results with the rigidity of the cytoskeleton of VSMCs. The results suggest that the alignment degree increases from peripheral arteries, such as carotid and femoral, to central arteries, as well coronary and thoracic aorta, which can indicate that the level of local alignment of the actin fibers in VSMCs is related with the mechanical behavior of the arterial wall. © 2018 International Society for Advancement of Cytometry.

Neurally Adjusted Ventilatory Assist (NAVA) or Pressure Support Ventilation (PSV) during spontaneous breathing trials in critically ill patients: a crossover trial.

BACKGROUND: Neurally Adjusted Ventilatory Assist (NAVA) is a proportional ventilatory mode that uses the electrical activity of the diaphragm (EAdi) to offer ventilatory assistance in proportion to patient effort. NAVA has been increasingly used for critically ill patients, but it has not been evaluated during spontaneous breathing trials (SBT). We designed a pilot trial to assess the feasibility of using NAVA during SBTs, and to compare the breathing pattern and patient-ventilator asynchrony of NAVA with Pressure Support (PSV) during SBTs. METHODS: We conducted a crossover trial in the ICU of a university hospital in Brazil and included mechanically ventilated patients considered ready to undergo an SBT on the day of the study. Patients underwent two SBTs in randomized order: 30 min in PSV of 5 cmH2O or NAVA titrated to generate equivalent peak airway pressure (Paw), with a positive end-expiratory pressure of 5 cmH2O. The ICU team, blinded to ventilatory mode, evaluated whether patients passed each SBT. We captured flow, Paw and electrical activity of the diaphragm (EAdi) from the ventilator and used it to calculate respiratory rate (RR), tidal volume (VT), and EAdi. Detection of asynchrony events used waveform analysis and we calculated the asynchrony index as the number of asynchrony events divided by the number of neural cycles. RESULTS: We included 20 patients in the study. All patients passed the SBT in PSV, and three failed the SBT in NAVA. Five patients were reintubated and the extubation failure rate was 25% (95% CI 9-49%). Respiratory parameters were similar in the two modes: VT = 6.1 (5.5-6.5) mL/Kg in NAVA vs. 5.5 (4.8-6.1) mL/Kg in PSV (p = 0.076) and RR = 27 (17-30) rpm in NAVA vs. 26 (20-30) rpm in PSV, p = 0.55. NAVA reduced AI, with a median of 11.5% (4.2-19.7) compared to 24.3% (6.3-34.3) in PSV (p = 0.033). CONCLUSIONS: NAVA reduces patient-ventilator asynchrony index and generates a respiratory pattern similar to PSV during SBTs. Patients considered ready for mechanical ventilation liberation may be submitted to an SBT in NAVA using the same objective criteria used for SBTs in PSV. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT01337271 ), registered April 12, 2011.

Monitoring the electric activity of the diaphragm during noninvasive positive pressure ventilation: a case report.

BACKGROUND: In patients with post-extubation respiratory distress, delayed reintubation may worsen clinical outcomes. Objective measures of extubation failure at the bedside are lacking, therefore clinical parameters are currently used to guide the need of reintubation. Electrical activity of the diaphragm (EAdi) provides clinicians with valuable, objective information about respiratory drive and could be used to monitor respiratory effort. CASE PRESENTATION: We describe the case of a patient with Chronic Obstructive Pulmonary Disease (COPD), from whom we recorded EAdi during four different ventilatory conditions: 1) invasive mechanical ventilation, 2) spontaneous breathing trial (SBT), 3) unassisted spontaneous breathing, and 4) Noninvasive Positive Pressure Ventilation (NPPV). The patient had been intubated due to an exacerbation of COPD, and after four days of mechanical ventilation, she passed the SBT and was extubated. Clinical signs of respiratory distress were present immediately after extubation, and EAdi increased compared to values obtained during mechanical ventilation. As we started NPPV, EAdi decreased substantially, indicating muscle unloading promoted by NPPV, and we used the EAdi signal to monitor respiratory effort during NPPV. Over the next three days, she was on NPPV for most of the time, with short periods of spontaneous breathing. EAdi remained considerably lower during NPPV than during spontaneous breathing, until the third day, when the difference was no longer clinically significant. She was then weaned from NPPV and discharged from the ICU a few days later. CONCLUSION: EAdi monitoring during NPPV provides an objective parameter of respiratory drive and respiratory muscle unloading and may be a useful tool to guide post-extubation ventilatory support. Clinical studies with continuous EAdi monitoring are necessary to clarify the meaning of its absolute values and changes over time.

Non-equilibrium cytoquake dynamics in cytoskeletal remodeling and stabilization.

The cytoskeleton (CSK) is a tensed fiber framework that supports, shapes and stabilizes the cell. The CSK is in a constant state of remodeling, moreover, which is an active non-equilibrium thermodynamic process. We report here that cytoskeletal remodeling involves reconfigurations that are not only sudden but also are transmitted to great distances within the cell in a fashion reminiscent of quakes in the Earth's crust. Remarkably, these events in the cell conform both qualitatively and quantitatively to empirical laws typical of earthquakes, including hierarchical fault structures, cumulative energy distributions following the Gutenberg-Richter law, and rate of after-shocks following Omori's law. While it is well-established that remodeling and stabilization of the cytoskeleton are non-equilibrium process, these new unanticipated observations establish that these processes are also remarkably non-local and strongly cooperative.

Diesel exhaust particulates affect cell signaling, mucin profiles, and apoptosis in trachea explants of Balb/C mice.

Particulate matter from diesel exhaust (DEP) has toxic properties and can activate intracellular signaling pathways and induce metabolic changes. This study was conducted to evaluate the activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and to analyze the mucin profile (acid (AB(+) ), neutral (PAS(+) ), or mixed (AB/PAS(+) ) mucus) and vacuolization (V) of tracheal explants after treatment with 50 or 100 µg/mL DEP for 30 or 60 min. Western blot analyses showed small increases in ERK1/2 and JNK phosphorylation after 30 min of 100 µg/mL DEP treatment compared with the control. An increase in JNK phosphorylation was observed after 60 min of treatment with 50 µg/mL DEP compared with the control. We did not observe any change in the level of ERK1/2 phosphorylation after treatment with 50 µg/mL DEP. Other groups of tracheas were subjected to histological sectioning and stained with periodic acid-Schiff (PAS) reagent and Alcian Blue (AB). The stained tissue sections were then subjected to morphometric analysis. The results obtained were compared using ANOVA. Treatment with 50 µg/mL DEP for 30 min or 60 min showed a significant increase (p < 0.001) in the amount of acid mucus, a reduction in neutral mucus, a significant reduction in mixed mucus, and greater vacuolization. Our results suggest that compounds found in DEPs are able to activate acid mucus production and enhance vacuolization and cell signaling pathways, which can lead to airway diseases.

Variation of mechanical properties and quantitative proteomics of VSMC along the arterial tree.

Vascular smooth muscle cells (VSMCs) are thought to assume a quiescent and homogeneous mechanical behavior after arterial tree development phase. However, VSMCs are known to be molecularly heterogeneous in other aspects and their mechanics may play a role in pathological situations. Our aim was to evaluate VSMCs from different arterial beds in terms of mechanics and proteomics, as well as investigate factors that may influence this phenotype. VSMCs obtained from seven arteries were studied using optical magnetic twisting cytometry (both in static state and after stretching) and shotgun proteomics. VSMC mechanical data were correlated with anatomical parameters and ultrastructural images of their vessels of origin. Femoral, renal, abdominal aorta, carotid, mammary, and thoracic aorta exhibited descending order of stiffness (G, P < 0.001). VSMC mechanical data correlated with the vessel percentage of elastin and amount of surrounding extracellular matrix (ECM), which decreased with the distance from the heart. After 48 h of stretching simulating regional blood flow of elastic arteries, VSMCs exhibited a reduction in basal rigidity. VSMCs from the thoracic aorta expressed a significantly higher amount of proteins related to cytoskeleton structure and organization vs. VSMCs from the femoral artery. VSMCs are heterogeneous in terms of mechanical properties and expression/organization of cytoskeleton proteins along the arterial tree. The mechanical phenotype correlates with the composition of ECM and can be modulated by cyclic stretching imposed on VSMCs by blood flow circumferential stress.

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes.

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

Cobalamin decyanation by the membrane transporter BtuM.

BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the ß-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the ß-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 µM), followed by the formation of a covalent bond (rate constant of 0.163 s-1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.

Random-walk model of the sodium-glucose transporter SGLT2 with stochastic steps and inhibition.

Random-walk models are frequently used to model distinct natural phenomena such as diffusion processes, stock-market fluctuations, and biological systems. Here, we present a random-walk model to describe the dynamics of glucose uptake by the sodium-glucose transporter of type 2, SGLT2. Our starting point is the canonical alternating-access model, which suggests the existence of six states for the transport cycle. We propose the inclusion of two new states to this canonical model. The first state is added to implement the recent discovery that the Na+ion can exit before the sugar is released into the proximal tubule epithelial cells. The resulting model is a seven-state mechanism with stochastic steps. Then we determined the transition probabilities between these seven states and used them to write a set of master equations to describe the time evolution of the system. We showed that our model converges to the expected equilibrium configuration and that the binding of Na+and glucose to SGLT2 in the inward-facing conformation must be necessarily unordered. After that, we added another state to implement inhibition in the model. Our results reproduce the experimental dependence of glucose uptake on the inhibitor concentration and they reveal that the inhibitors act by decreasing the number of available SGLT2s, which increases the chances of glucose escaping reabsorption.

Mapping the cytoskeletal prestress.

Cell mechanical properties on a whole cell basis have been widely studied, whereas local intracellular variations have been less well characterized and are poorly understood. To fill this gap, here we provide detailed intracellular maps of regional cytoskeleton (CSK) stiffness, loss tangent, and rate of structural rearrangements, as well as their relationships to the underlying regional F-actin density and the local cytoskeletal prestress. In the human airway smooth muscle cell, we used micropatterning to minimize geometric variation. We measured the local cell stiffness and loss tangent with optical magnetic twisting cytometry and the local rate of CSK remodeling with spontaneous displacements of a CSK-bound bead. We also measured traction distributions with traction microscopy and cell geometry with atomic force microscopy. On the basis of these experimental observations, we used finite element methods to map for the first time the regional distribution of intracellular prestress. Compared with the cell center or edges, cell corners were systematically stiffer and more fluidlike and supported higher traction forces, and at the same time had slower remodeling dynamics. Local remodeling dynamics had a close inverse relationship with local cell stiffness. The principal finding, however, is that systematic regional variations of CSK stiffness correlated only poorly with regional F-actin density but strongly and linearly with the regional prestress. Taken together, these findings in the intact cell comprise the most comprehensive characterization to date of regional variations of cytoskeletal mechanical properties and their determinants.

Modified 3D Ewald Summation for Slab Geometry at Constant Potential.

We present a new Monte Carlo method to simulate ionic liquids in slab geometry at constant potential. The algorithm is built upon two previous methods while retaining the advantages of each of them. The method is tested against a Poisson-Boltzmann theory and the constant surface charge ensemble, achieving consistency among all of them. We then analyze the computational time of the developed algorithm, showing substantial speedup in relation to the method of Kiyohara and Asaka [J. Chem. Phys., 2007, 126, 214704]. As an application of our method, we investigate crowding and overscreening in confined room-temperature ionic liquids. We show that we can switch between two behaviors of the double layer by changing the Bjerrum length alone.

Random-walk model of cotransport.

We present a statistical mechanical model to describe the dynamics of an arbitrary cotransport system. Our starting point was the alternating access mechanism, which suggests the existence of six states for the cotransport cycle. Then we determined the 14 transition probabilities between these states, including a leak pathway, and used them to write a set of Master Equations for describing the time evolution of the system. The agreement between the asymptotic behavior of this set of equations and the result obtained from thermodynamics is a confirmation that leakage is compatible with the static head equilibrium condition and that our model has captured the essential physics of cotransport. In addition, the model correctly reproduced the transport dynamics found in the literature.

Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples.

Knockout of multifunction gene cysteine- and glycine-rich protein 3 (CSRP3) in cardiomyocytes (CMs) of mice leads to heart dilation, severely affecting its functions. In humans, CSRP3 mutations are associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM). The absence of the CSRP3 expression produces unknown effects on in vitro neonatal CMs' metabolism. The metabolome changes in culture media conditioned by CSRP3 knockout (KO-CSRP3), and wild type (WT) neonatal cardiomyocytes were investigated under untreated or after metabolic challenging conditions produced by isoproterenol (ISO) stimulation, by in vitro high-resolution proton magnetic resonance spectroscopy (1H-MRS)-based metabolomics. Metabolic differences between neonatal KO-CSRP3 and WT rats' CMs were identified. After 72 h of culture, ISO administration was associated with increased CMs' energy requirements and increased levels of threonine, alanine, and 3-hydroxybutyrate in both neonatal KO-CSRP3 and WT CMs conditioned media. When compared with KO-CSRP3, culture media derived from WT cells presented higher lactate concentrations either under basal or ISO-stimulated conditions. The higher activity of ketogenic biochemical pathways met the elevated energy requirements of the contractile cells. Both cells are considered phenotypically indistinguishable in the neonatal period of animal lives, but the observed metabolic stress responses of KO-CSRP3 and WT CMs to ISO were different. KO-CSRP3 CMs produced less lactate than WT CMs in both basal and stimulated conditions. Mainly, ISO-stimulated conditions produced evidence for lactate overload within KO-CSRP3 CMs, while WT CMs succeeded to manage the metabolic stress. Thus, 1H-MRS-based metabolomics was suitable to identify early inefficient energetic metabolism in neonatal KO-CSRP3 CMs. These results may reflect an apparent lower lactate transport and consumption, in association with protein catabolism.

Neurally adjusted ventilatory assist vs. pressure support to deliver protective mechanical ventilation in patients with acute respiratory distress syndrome: a randomized crossover trial.

BACKGROUND: Protective mechanical ventilation is recommended for patients with acute respiratory distress syndrome (ARDS), but it usually requires controlled ventilation and sedation. Using neurally adjusted ventilatory assist (NAVA) or pressure support ventilation (PSV) could have additional benefits, including the use of lower sedative doses, improved patient-ventilator interaction and shortened duration of mechanical ventilation. We designed a pilot study to assess the feasibility of keeping tidal volume (VT) at protective levels with NAVA and PSV in patients with ARDS. METHODS: We conducted a prospective randomized crossover trial in five ICUs from a university hospital in Brazil and included patients with ARDS transitioning from controlled ventilation to partial ventilatory support. NAVA and PSV were applied in random order, for 15 min each, followed by 3 h in NAVA. Flow, peak airway pressure (Paw) and electrical activity of the diaphragm (EAdi) were captured from the ventilator, and a software (Matlab, Mathworks, USA), automatically detected inspiratory efforts and calculated respiratory rate (RR) and VT. Asynchrony events detection was based on waveform analysis. RESULTS: We randomized 20 patients, but the protocol was interrupted for five (25%) patients for whom we were unable to maintain VT below 6.5 mL/kg in PSV due to strong inspiratory efforts and for one patient for whom we could not detect EAdi signal. For the 14 patients who completed the protocol, VT was 5.8 ± 1.1 mL/kg for NAVA and 5.6 ± 1.0 mL/kg for PSV (p = 0.455) and there were no differences in RR (24 ± 7 for NAVA and 23 ± 7 for PSV, p = 0.661). Paw was greater in NAVA (21 ± 3 cmH2O) than in PSV (19 ± 3 cmH2O, p = 0.001). Most patients were under continuous sedation during the study. NAVA reduced triggering delay compared to PSV (p = 0.020) and the median asynchrony Index was 0.7% (0-2.7) in PSV and 0% (0-2.2) in NAVA (p = 0.6835). CONCLUSIONS: It was feasible to keep VT in protective levels with NAVA and PSV for 75% of the patients. NAVA resulted in similar VT, RR and Paw compared to PSV. Our findings suggest that partial ventilatory assistance with NAVA and PSV is feasible as a protective ventilation strategy in selected ARDS patients under continuous sedation. Trial registration ClinicalTrials.gov (NCT01519258). Registered 26 January 2012, https://clinicaltrials.gov/ct2/show/NCT01519258.

Alterations in cellular force parameters and cell projections in Nasal polyps-derived fibroblasts.

OBJECTIVE: Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) is a disease that features a mechanical dysfunction involving chronic inflammation and altered tissue remodeling. In this study, we aim to evaluate the fibroblast morphology and its cellular traction force in primary fibroblasts cell cultures obtained from both healthy individuals (n=7) and patients with CRSwNP (n=8). METHODS: Using a Traction-force Microscopy we analyzed parameters of Force/Tension in fibroblasts cultures in both experimental groups. RESULTS: The analysis of the Projected Area of Cell revealed that fibroblasts derived from nasal mucosa of healthy individuals have an area on average 39.24% larger than the fibroblasts obtained from the nasal polyp tissue. We also observed that the parameters directly related to the force of the cell, Max Cumulative Force and Net Contractile Moment, presented a high Force/Tension per unit of area in the fibroblasts derived from the healthy nasal mucosa (on average 41% and 52.54% higher than the fibroblasts of the nasal polyp respectively). CONCLUSION: Our results demonstrate a cellular mechanism that may be associated with the mechanical dysfunction found in the Nasal Polyp tissue. The weak traction force of nasal polyp-derived fibroblast may, in lower dimensions, impact on the remodeling of nasal mucosa in CRSwNP.

Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice.

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.

Thermodynamic origin of cooperativity in actomyosin interactions: the coupling of short-range interactions with actin bending stiffness in an Ising-like model.

We present Monte Carlo simulations for a molecular motor system found in virtually all eukaryotic cells, the acto-myosin motor system, composed of a group of organic macromolecules. Cell motors were mapped to an Ising-like model, where the interaction field is transmitted through a tropomyosin polymer chain. The presence of Ca2+ induces tropomyosin to block or unblock binding sites of the myosin motor leading to its activation or deactivation. We used the Metropolis algorithm to find the transient and the equilibrium states of the acto-myosin system composed of solvent, actin, tropomyosin, troponin, Ca2+, and myosin-S1 at a given temperature, including the spatial configuration of tropomyosin on the actin filament surface. Our model describes the short- and long-range cooperativity during actin-myosin binding which emerges from the bending stiffness of the tropomyosin complex. We found all transition rates between the states only using the interaction energy of the constituents. The agreement between our model and experimental data also supports the recent theory of flexible tropomyosin.