Fernando Antunes

High-level bio-decontamination, which involves reducing microorganisms by 99.9999%, is essential in preventing Hospital Acquired Infections and controlling pandemics. This study demonstrates that nanoporous materials, which can retain molecules within their pores, and subsequently release them, can be used in high level bio-decontamination. H2O2 vapor is a golden-standard in high level bio-decontamination. This work demonstrates that various nanoporous materials, particularly mesoporous silicas, can be utilized to store and release H2O2 in the vapor phase. H2O2 concentrations of over 2500 ppm were achieved by desorbing it from the carrier material at low temperatures of 60-80 °C. Generation of H2O2-vapor by desorption from nanoporous materials is technically much simpler than vaporization of aqueous H2O2 solutions, which use flash vaporization processes occurring at 130-150 °C. This has important technical implications, highlighting the potential of nanoporous materials as carriers for H2O2 for high-level bio-decontamination.

Hydrogen peroxide (H₂O₂) plays a central role in cellular signaling, but its signaling mechanisms are just beginning to be understood. Here we analyze and review the evidence about the spatial distribution of H₂O₂ in cells and its implications for redox relays, H₂O₂ channeling, and other redox signaling mechanisms. Altogether, theoretical and experimental approaches consistently support the conclusions that (1) basal mean cytosolic H₂O₂ concentrations in eukaryotic cells do not exceed the low-nanomolar range; (2) at low oxidative loads cytosolic H₂O₂ is confined to microdomains; (3) transmembrane gradients in cells exposed to sub-micromolar H₂O₂ are at least in the mid-hundredfold scale; (4) the low cytosolic H₂O₂ concentrations, their uneven spatial distribution, and the strong transmembrane gradients are mainly a consequence of the action of the peroxiredoxin/thioredoxin/thioredoxin reductase system; (5) the efficacy of the signal transmission by redox switches is modulated by their oxidation and reduction; (6) in the cytosol, signaling at low oxidative loads proceeds mainly by the formation of local microdomains, where oxidized forms of peroxiredoxins are also concentrated; and (7) in cellular compartments with higher levels of H₂O₂, like the endoplasmic reticulum, channeling H₂O₂ directly to the target is expected to constitute an important mechanism underlying signal transmission.

The synthesis and scavenging of hydrogen peroxide (H₂O₂) is tightly regulated in all cell types. H₂O₂ can fluctuate from low, physiological levels to intermediate, signaling ones and can further raise to participate in toxic reactions. Cell membranes contribute to the generation of gradients between the extracellular and intracellular media, as well as between cellular compartments. Elucidating steady-state levels of H₂O₂ at different cell compartments and the gradients across membranes is essential to understand redox signaling and toxicity effects. The most efficient tools for specific, quantitative, spatial, and timely measurements of H₂O₂ are genetically encoded biosensors of two families: redox-sensitive (ro)GFP fused to peroxiredoxins and HyPer derivatives. We will briefly describe here the advantages of three members of these families (roGFP2-Tpx1.C169S, HyPer, and Hyper7) and how the application of mathematical equations to the experimental data obtained with these probes has proven very useful to determine peroxide gradients across membranes, and can be used for calculating absolute and relative steady-state levels of H₂O₂ in different eukaryotic models.

SUMMARY: Metabolic alterations have been recognized to underly the etiology of many diseases. Herein, cellular energy dissipation was evaluated as a novel non-specific global biomarker of metabolic alterations. Energy dissipation, measured as heat by microcalorimetry, was maximal during Saccharomyces cerevisiae adaptation to growth conditions before fast proliferation took place. This response was further augmented by 95 % in media where nutrient assimilation was more difficult, and by 133 % under sub-optimal non-carbon nutrient levels. In this last case, the increase in energy dissipation (1) reflected changes in amino acid and glycolytic metabolism and (2) anticipated changes in the growth curve significantly later observed by traditional microbiological measurements. It was, therefore, an early marker of adaptive responses that compensated for sub-optimal nutrient levels and maintained phenotypic stability. Compensatory responses buffer systems against perturbations and delay the onset of diseases. Microcalorimetry can, therefore, provide a biomarker development platform for early disease-diagnosis. HIGHLIGHTS: Energy dissipation measurements detect cell responses to metabolic challenges. The detection by microcalorimetry occurs considerably earlier than by traditional microbiological measurements. Sub-optimal non-carbon nutrient levels impact energy dissipation long before cell proliferation. Energy dissipation is highly sensitive to increased nutrient assimilation difficulty.

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.

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.

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.