Ultra-Microporous Fe-MOF with Prolonged NO Delivery in Biological Media for Therapeutic Application.

Nitric oxide (NO), a key element in the regulation of essential biological mechanisms, presents huge potential as therapeutic agent in the treatment and prevention of chronic diseases. Metal-organic frameworks (MOFs) with open metal sites are promising carriers for NO therapies but delivering it over an extended period in biological media remains a great challenge due to i) a fast degradation of the material in body fluids and/or ii) a rapid replacement of NO by water molecules onto the Lewis acid sites. Here, a new ultra-narrow pores Fe bisphosphonate MOF, denoted MIP-210(Fe) or Fe(H2O)(Hmbpa) (H4mbpa = p-xylenediphosphonic acid) is described that adsorbs NO due to an unprecedented sorption mechanism: coordination of NO through the Fe(III) sites is unusually preferred, replacing bound water, and creating a stable interaction with the free H2O and P-OH groups delimiting the ultra-narrow pores. This, associated with the high chemical stability of the MOF in body fluids, enables an unprecedented slow replacement of NO by water molecules in biological media, achieving an extraordinarily extended NO delivery time over at least 70 h, exceeding by far the NO kinetics release reported with others porous materials, paving the way for the development of safe and successful gas therapies.

Wheel Slip Control Applied to an Electric Tractor for Improving Tractive Efficiency and Reducing Energy Consumption.

This work presents an automatic slip control solution applied to a two-wheel-drive (2WD) electric tractor. Considering that the slip can be maintained within a specific range that depends on the type of soil, it is possible to increase the tractive efficiency of the electric vehicle (EV). The control system can be easily designed considering only the longitudinal dynamics of the tractor while using simple proportional-integral (PI) controllers to drive the inverters associated with the rear wheels. The introduced solution is tested on an experimental electric tractor prototype traveling on firm soil considering case studies in which the slip control is enabled and disabled. The acquired results demonstrate that the slip control allows for obtaining a more stable performance and reduced energy consumption.

A Comparison of Different Approaches to Quantify Nitric Oxide Release from NO-Releasing Materials in Relevant Biological Media.

The development of solid materials that deliver nitric oxide (NO) are of interest for several therapeutic applications. Nevertheless, due to NO's reactive nature, rapid diffusion and short half-life, reporting their NO delivery characteristics is rather complex. The full knowledge of this parameter is fundamental to discuss the therapeutic utility of these materials, and thus, the NO quantification strategy must be carefully considered according to the NO-releasing scaffold type, to the expected NO-releasing amounts and to the medium of quantification. In this work, we explore and discuss three different ways of quantifying the release of NO in different biological fluids: haemoglobin assay, Griess assay and NO electrochemical detection. For these measurements, different porous materials, namely zeolites and titanosilicates were used as models for NO-releasing platforms. The oxyhaemoglobin assay offers great sensitivity (nanomolar levels), but it is only possible to monitor the NO release while oxyhaemoglobin is not fully converted. On the other hand, Griess assay has low sensitivity in complex biological media, namely in blood, and interferences with media make NO measurements questionable. Nevertheless, this method can measure micromolar amounts of NO and may be useful for an initial screening for long-term release performance. The electrochemical sensor enabled real-time measurements in a variety of biological settings. However, measured NO is critically low in oxygenated and complex media, giving transient signals, which makes long-term quantification impossible. Despite the disadvantages of each method, the combination of all the results provided a more comprehensive NO release profile for these materials, which will help to determine which formulations are most promising for specific therapeutic applications. This study highlights the importance of using appropriate NO quantification tools to provide accurate reports.

Tuning Cellular Biological Functions Through the Controlled Release of NO from a Porous Ti-MOF.

Materials for the controlled release of nitric oxide (NO) are of interest for therapeutic applications. However, to date, many suffer from toxicity and stability issues, as well as poor performance. Herein, we propose a new NO adsorption/release mechanism through the formation of nitrites on the skeleton of a titanium-based metal-organic framework (MOF) that we named MIP-177, featuring a suitable set of properties for such an application: (i) high NO storage capacity (3 µmol mg-1solid ), (ii) excellent biocompatibility at therapeutic relevant concentrations (no cytotoxicity at 90 µg mL-1 for wound healing) due to its high stability in biological media (<9 % degradation in 72 hours) and (iii) slow NO release in biological media (≈2 hours for 90 % release). The prospective application of MIP-177 is demonstrated through NO-driven control of mitochondrial respiration in cells and stimulation of cell migration, paving the way for the design of new NO delivery systems for wound healing therapy.

New generation of nitric oxide-releasing porous materials: Assessment of their potential to regulate biological functions.

Nitric oxide (NO) presents innumerable biological roles, and its exogenous supplementation for therapeutic purposes has become a necessity. Some nanoporous materials proved to be potential vehicles for NO with high storage capacity. However, there is still a lack of information about their efficiency to release controlled NO and if they are biocompatible and biologically stable. In this work, we address this knowledge gap starting by evaluating the NO release and stability under biological conditions and their toxicity with primary keratinocyte cells. Titanosilicates (ETS-4 and ETS-10 types) and clay-based materials were the materials under study, which have shown in previous studies suitable NO gas adsorption/release rates. ETS-4 proved to be the most promising material, combining good biocompatibility at 180 µg/mL, stability and slower NO release. ETS-10 and ETAS-10 showed the best biocompatibility at the same concentration and, in the case of clay-based materials, CoOS is the least toxic of those tested and the one that releases the highest NO amount. The potentiality of these new NO donors to regulate biological functions was assessed next by controlling the mitochondrial respiration and the cell migration. NO-loaded ETS-4 regulates O2 consumption and cell migration in a dose-dependent manner. For cell migration, a biphasic effect was observed in a narrow range of ETS-4 concentration, with a stimulatory effect becoming inhibitory just by doubling ETS-4 concentration. For the other materials, no effective regulation was achieved, which highlights the relevance of the new assessment presented in this work for nanoporous NO carriers that will pave the way for further developments.

Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity.

BACKGROUND: Hydrogen peroxide (H2O2) is generated as a by-product of metabolic reactions during oxygen use by aerobic organisms, and can be toxic or participate in signaling processes. Cells, therefore, need to be able to sense and respond to H2O2 in an appropriate manner. This is often accomplished through thiol switches: Cysteine residues in proteins that can act as sensors, and which are both scarce and finely tuned. Bacteria and eukaryotes use different types of such sensors-either a one-component (OxyR) or two-component (Pap1-Tpx1) redox relay, respectively. However, the biological significance of these two different signaling modes is not fully understood, and the concentrations and peroxides driving those types of redox cascades have not been determined, nor the intracellular H2O2 levels linked to toxicity. Here we elucidate the characteristics, rates, and dynamic ranges of both systems. RESULTS: By comparing the activation of both systems in fission yeast, and applying mathematical equations to the experimental data, we estimate the toxic threshold of intracellular H2O2 able to halt aerobic growth, and the temporal gradients of extracellular to intracellular peroxides. By calculating both the oxidation rates of OxyR and Tpx1 by peroxides, and their reduction rates by the cellular redoxin systems, we propose that, while Tpx1 is a sensor and an efficient H2O2 scavenger because it displays fast oxidation and reduction rates, OxyR is strictly a H2O2 sensor, since its reduction kinetics are significantly slower than its oxidation by peroxides, and therefore, it remains oxidized long enough to execute its transcriptional role. We also show that these two paradigmatic H2O2-sensing models are biologically similar at pre-toxic peroxide levels, but display strikingly different activation behaviors at toxic doses. CONCLUSIONS: Both Tpx1 and OxyR contain thiol switches, with very high reactivity towards peroxides. Nevertheless, the fast reduction of Tpx1 defines it as a scavenger, and this efficient recycling dramatically changes the Tpx1-Pap1 response to H2O2 and connects H2O2 sensing to the redox state of the cell. In contrast, OxyR is a true H2O2 sensor but not a scavenger, being partially insulated from the cellular electron donor capacity.

Rat Aquaporin-5 Is pH-Gated Induced by Phosphorylation and Is Implicated in Oxidative Stress.

Aquaporin-5 (AQP5) is a membrane water channel widely distributed in human tissues that was found up-regulated in different tumors and considered implicated in carcinogenesis in different organs and systems. Despite its wide distribution pattern and physiological importance, AQP5 short-term regulation was not reported and mechanisms underlying its involvement in cancer are not well defined. In this work, we expressed rat AQP5 in yeast and investigated mechanisms of gating, as well as AQP5's ability to facilitate H2O2 plasma membrane diffusion. We found that AQP5 can be gated by extracellular pH in a phosphorylation-dependent manner, with higher activity at physiological pH 7.4. Moreover, similar to other mammalian AQPs, AQP5 is able to increase extracellular H2O2 influx and to affect oxidative cell response with dual effects: whereas in acute oxidative stress conditions AQP5 induces an initial higher sensitivity, in chronic stress AQP5 expressing cells show improved cell survival and resistance. Our findings support the involvement of AQP5 in oxidative stress and suggest AQP5 modulation by phosphorylation as a novel tool for therapeutics.

Potential antitumour and pro-oxidative effects of (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR).

CONTEXT: Characterization of the pro-oxidant activity of QNACR. OBJECTIVES: Reactive oxygen species (ROS) induce cellular damage and represent unique opportunities to kill malignant cells. In this study, we synthesized and evaluated the new compound, (E)-methyl 2-(7-chloroquinolin-4-ylthio)-3-(4-hydroxyphenyl) acrylate (QNACR) as potential pro-oxidative agent against breast cancer. METHODS: Oxidative stress biomarkers such as ROS, thiobarbuturic acid reactive species (TBARs) and different antioxidant enzyme activities were determined in cell lysates. RESULTS: QNACR showed cytotoxic and more selective effects to tumour MCF7 cells (IC50 < 25 µM) compared to antitumour controls, inducing ROS and TBARs parallel to inhibitions of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Longer exposures to QNACR triggered adaptive effects increasing the overall activities of CAT, glutathione reductase, G6PDH and 6PGDH, but eventually the adaptation changes faded and cells died. CONCLUSION: QNACR led to remarkable modifications in the oxidative status of tumour cells, proposing this compound as potential alternative for antitumour therapy.

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts.

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

Theoretical analysis of the kinetic performance of laboratory- and full-scale composting systems.

Composting research at laboratory-scale is critical for the development of optimized full-scale plants. Discrepancies between processes at laboratory-scale and full-scale systems have been investigated in terms of heat balances, but a kinetic analysis of this issue is still missing. In this study, the composting rate at laboratory-scale was, on average, between 1.9 and 5.7 times faster than in full-scale systems for a set of published studies using municipal solid waste, food waste or similar materials. Laboratory-scale performance and full-scale systems were limited to 71 and 46%, respectively, of their maximum potential due to poor management of environmental process conditions far from their optimum. The main limiting environmental factor was found to be moisture content, followed by temperature. Besides environmental factors, waste composition and particle size were identified as factors accounting for kinetic differences between laboratory- and full-scale systems. Overall, this study identifies those factors that affect the kinetics of the composting process most and revealed a significant margin for reducing process time in full-scale composting.

Fatigue Crack Growth in Metallic Materials.

Mechanical components and structures are submitted to cyclic loads in different applications; therefore, they must be designed to withstand fatigue [...].

Data Processing to Probe the Cellular Hydrogen Peroxide Landscape.

Hydrogen peroxide (H2O2) regulates signaling pathways by modulating the activity of redox-sensitive proteins denominated redox switches. The magnitude of the transient variations in localized H2O2 pools during signaling events and how these variations impact redox switches present in the cell remain elusive. A canonical model with two chemical reactions comprising the oxidation/reduction cycle of a redox switch is described. The model is dimensionless with respect to the redox switch concentration. Thus, the time-series data required to apply the equations deduced is the percentage of oxidation of a redox switch, avoiding the application of absolute concentrations that are often difficult to measure experimentally. Here, we describe detailed protocols for the processing of experimental data with the canonical model to probe the absolute concentrations of H2O2 found in the vicinity of redox switches and probes, as well as the kinetic parameters that describe the reduction and oxidation of redox switches. The protocols are an analytical tool that helps to depict the cellular hydrogen peroxide signaling landscape, giving new insights on H2O2 signaling mechanisms, and hold the potential to be a framework for a future redox kinetomics analytical platform.

Emerging Nitric Oxide and Hydrogen Sulfide Releasing Carriers for Skin Wound Healing Therapy.

Nitric oxide (NO) and hydrogen sulfide (H2 S) have been recognized as important signalling molecules involved in multiple physiological functions, including wound healing. Their exogenous delivery has been established as a new route for therapies, being the topical application the nearest to commercialization. Nevertheless, the gaseous nature of these therapeutic agents and their toxicity at high levels imply additional challenges in the design of effective delivery systems, including the tailoring of their morphology and surface chemistry to get controllable release kinetics and suitable lifetimes. This review highlights the increasing interest in the use of these gases in wound healing applications by presenting the various potential strategies in which NO and/or H2 S are the main therapeutic agents, with focus on their conceptual design, release behaviour and therapeutic performance. These strategies comprise the application of several types of nanoparticles, polymers, porous materials, and composites as new releasing carriers of NO and H2 S, with characteristics that will facilitate the application of these molecules in the clinical practice.

Effect of Residual Stresses on Fatigue Crack Growth: A Numerical Study Based on Cumulative Plastic Strain at the Crack Tip.

Residual stresses affect the fatigue behavior, given that compressive stresses delay the phenomenon, while tensile stresses accelerate it. However, the mechanisms behind the effect of residual stresses are not totally understood. A numerical study is developed here to understand the effect of thermal residual stresses (TRSs) on fatigue crack growth (FCG). The crack driving force was assumed to be the cumulative plastic strain at the crack tip. The heating of a region ahead of the crack tip produced elastic compressive TRS, which were 69% of material's yield stress. Alternatively, plastic deformation was produced by severe cooling followed by heating to generate compressive residual stresses. The crack propagation in the compressive residual stress field produced a decrease in the FCG rate. On the other hand, without the contact of crack flanks, the TRS showed no effect on FCG. Therefore, the TRSs only affect FCG by changing the crack closure level.

Biphasic modulation of fatty acid synthase by hydrogen peroxide in Saccharomyces cerevisiae.

Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150µM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit ß. However, the exposure to a steady-state 50µM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.

Diagnosis and optimization of the composting process in full-scale mechanical-biological treatment plants.

The aims of this study were (i) to evaluate the performance of the composting process operation in full-scale mechanical-biological treatment (MBT) plants, (ii) to estimate their performance under optimized conditions and (iii) to propose specific guidelines on how to improve the efficiency of the composting process. To fulfil these objectives, a first-order kinetic model was used. This model was calibrated with experimental data to account for the limitations imposed by less-than-optimal environmental conditions during operation of the composting process. Data treatment and simulation showed that two of the three MBT plants studied were poorly operated. Optimization of process management with measures of simple practical implementation was estimated to be highly significant in these poorly managed plants, increasing performance by 103% in MBT1 and 53% in MBT2. In MBT3, the potential for optimization was estimated at 17%. Similar results were obtained from the analysis of other published data, suggesting that poor process management in MBT composting is widespread. These findings highlight the importance of having programmes for monitoring and optimizing process performance in full-scale composting systems. The procedures developed here are simple to apply and can routinely be implemented in full-scale plants.

Chitosan Biocomposites for the Adsorption and Release of H2S.

The search for H2S donors has been increasing due to the multiple therapeutic effects of the gas. However, the use of nanoporous materials has not been investigated despite their potential. Zeolites and activated carbons are known as good gas adsorbents and their modification with chitosan may increase the material biocompatibility and simultaneously its release time in aqueous solution, thus making them good H2S donors. Herein, we modified with chitosan a series of A zeolites (3A, 4A and 5A) with different pore sizes and an activated carbon obtained from glycerin. The amount of H2S adsorbed was evaluated by a volumetric method and their release capacity in aqueous solution was measured. These studies aimed to verify which of the materials had appropriate H2S adsorption/release properties to be considered a potential H2S donor. Additionally, cytotoxicity assays using HeLa cells were performed. Considering the obtained results, the chitosan composite with the A zeolite with the larger pore opening was the most promising material to be used as a H2S donor so a further cytotoxicity assay using H2S loaded was conducted and no toxicity was observed.

FCG Modelling Considering the Combined Effects of Cyclic Plastic Deformation and Growth of Micro-Voids.

Fatigue is one of the most prevalent mechanisms of failure. Thus, the evaluation of the fatigue crack growth process is fundamental in engineering applications subjected to cyclic loads. The fatigue crack growth rate is usually accessed through the da/dN-ΔK curves, which have some well-known limitations. In this study a numerical model that uses the cyclic plastic strain at the crack tip to predict da/dN was coupled with the Gurson-Tvergaard-Needleman (GTN) damage model. The crack propagation process occurs, by node release, when the cumulative plastic strain reaches a critical value. The GTN model is used to account for the material degradation due to the growth of micro-voids process, which affects fatigue crack growth. Predictions with GTN are compared with the ones obtained without this ductile fracture model. Crack closure was studied in order to justify the lower values of da/dN obtained in the model with GTN, when compared with the results without GTN, for lower ΔK values. Finally, the accuracy of both variants of the numerical model is accessed through the comparison with experimental results.

Glyceraldehyde-3-phosphate dehydrogenase is largely unresponsive to low regulatory levels of hydrogen peroxide in Saccharomyces cerevisiae.

BACKGROUND: The reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H2O2) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H2O2 in a variety of cellular systems. In yeast, when using a high initial H2O2 dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H2O2 functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H2O2 doses on GAPDH activity and expression in Saccharomyces cerevisiae. RESULTS: A calibrated and controlled method of H2O2 delivery - the steady-state titration - in which cells are exposed to constant, low, and known H2O2 concentrations, was used in this study. This technique, contrary to the common bolus addition, allows determining which H2O2 concentrations trigger specific biological responses. This work shows that both in exponential- and stationary-phase cells, low regulatory H2O2 concentrations induce a large upregulation of catalase, a fingerprint of the cellular oxidative stress response, but GAPDH oxidation and the ensuing activity decrease are only observed at death-inducing high H2O2 doses. GAPDH activity is constant upon incubation with sub-lethal H2O2 doses, but in stationary-phase cells there is a differential response in the expression of the three GAPDH isoenzymes: Tdh1p is strongly upregulated while Tdh2p/Tdh3p are slightly downregulated. CONCLUSIONS: In yeast GAPDH activity is largely unresponsive to low to moderate H2O2 doses. This points to a scenario where (a) cellular redoxins efficiently cope with levels of GAPDH oxidation induced by a vast range of sub-lethal H2O2 concentrations, (b) inactivation of GAPDH cannot be considered a sensitive biomarker of H2O2-induced oxidation in vivo. Since GAPDH inactivation only occurs at cell death-inducing high H2O2 doses, GAPDH-dependent rerouting of carbohydrate flux is probably important merely in pathophysiological situations. This work highlights the importance of studying H2O2-induced oxidative stress using concentrations closer to the physiological for determining the importance of protein oxidation phenomena in the regulation of cellular metabolism.

Revisiting Classical Issues of Fatigue Crack Growth Using a Non-Linear Approach.

Fatigue crack growth (FCG) has been studied for decades; however, several aspects are still objects of controversy. The objective here is to discuss different issues, using a numerical approach based on crack tip plastic strain, assuming that FCG is driven by crack tip deformation. ΔK was found to control cyclic plastic deformation at the crack tip, while Kmax has no effect. Therefore, alternative mechanisms are required to justify models based on ΔK and Kmax. The analysis of crack tip plastic deformation also showed that there is crack tip damage below crack closure. Therefore, the definition of an effective load range ΔKeff = Kmax - Kopen is not correct, because the portion of load range below opening also contributes to FCG. Below crack closure, damage occurs during unloading while during loading the crack tip deformation is elastic. However, if the maximum load is decreased below the elastic limit, which corresponds to the transition between elastic and elasto-plastic regimes, there is no crack tip damage. Additionally, a significant effect of the crack ligament on crack closure was found in tests with different crack lengths and the same ΔK. Finally, the analysis of FCG after an overload with and without contact of crack flanks showed that the typical variation of da/dN observed is linked to crack closure variations, while the residual stresses ahead of crack tip are not affected by the contact of crack flanks.