Hydropersulfides (RSSH) and Nitric Oxide (NO) Signaling: Possible Effects on S-Nitrosothiols (RS-NO)

S-Nitrosothiol (RS-NO) formation in proteins and peptides have been implicated as factors in the etiology of many diseases and as possible regulators of thiol protein function. They have also been proposed as possible storage forms of nitric oxide (NO). However, despite their proposed functions/roles, there appears to be little consensus regarding the physiological mechanisms of RS-NO formation and degradation. Hydropersulfides (RSSH) have recently been discovered as endogenously generated species with unique reactivity. One important reaction of RSSH is with RS-NO, which leads to the degradation of RS-NO as well as the release of NO. Thus, it can be speculated that RSSH can be a factor in the regulation of steady-state RS-NO levels, and therefore may be important in RS-NO (patho)physiology. Moreover, RSSH-mediated NO release from RS-NO may be a possible mechanism allowing RS-NO to serve as a storage form of NO.

Polydopamine-Assisted Surface Modification of Ti-6Al-4V Alloy with Anti-Biofilm Activity for Dental Implantology Applications

Coating the surfaces of implantable materials with various active principles to ensure inhibition of microbial adhesion, is a solution to reduce infections associated with dental implant. The aim of the study was to optimize the polydopamine films coating on the Ti-6Al-6V alloy surface in order to obtain a maximum of antimicrobial/antibiofilm efficacy and reduced cytotoxicity. Surface characterization was performed by evaluating the morphology (SEM, AFM) and structures (Solid-state 13C NMR and EPR). Antimicrobial activity was assessed by logarithmic reduction of CFU/mL, and the antibiofilm activity by reducing the adhesion of Escherichia coli, Staphylococcus aureus, and Candida albicans strains. The release of NO was observed especially for C. albicans strain, which confirms the results obtained for microbial adhesion. Among the PDA coatings, for 0.45:0.88 (KMnO4:dopamine) molar ratio the optimal compromise was obtained in terms of antimicrobial activity and cytotoxicity, while the 0.1:1.5 ratio (KMnO4:dopamine) led to higher NO release and implicitly the reduction of the adhesion capacities only for C. albicans, being slightly cytotoxic but with moderate release of LDH. The proposed materials can be used to reduce the adhesion of yeast to the implantable material and thus inhibit the formation of microbial biofilms.

Delivery of Nitric Oxide in the Cardiovascular System: Implications for Clinical Diagnosis and Therapy

Nitric oxide (NO) is a key molecule in cardiovascular homeostasis and its abnormal delivery is highly associated with the occurrence and development of cardiovascular disease (CVD). The assessment and manipulation of NO delivery is crucial to the diagnosis and therapy of CVD, such as endothelial dysfunction, atherosclerotic progression, pulmonary hypertension, and cardiovascular manifestations of coronavirus (COVID-19). However, due to the low concentration and fast reaction characteristics of NO in the cardiovascular system, clinical applications centered on NO delivery are challenging. In this tutorial review, we first summarized the methods to estimate the in vivo NO delivery process, based on computational modeling and flow-mediated dilation, to assess endothelial function and vulnerability of atherosclerotic plaque. Then, emerging bioimaging technologies that have the potential to experimentally measure arterial NO concentration were discussed, including Raman spectroscopy and electrochemical sensors. In addition to diagnostic methods, therapies aimed at controlling NO delivery to regulate CVD were reviewed, including the NO release platform to treat endothelial dysfunction and atherosclerosis and inhaled NO therapy to treat pulmonary hypertension and COVID-19. Two potential methods to improve the effectiveness of existing NO therapy were also discussed, including the combination of NO release platform and computational modeling, and stem cell therapy, which currently remains at the laboratory stage but has clinical potential for the treatment of CVD.

Removal of Antimony(V) from Drinking Water Using nZVI/AC: Optimization of Batch and Fix Bed Conditions

Antimony (Sb) traces in water pose a serious threat to human health due to their negative effects. In this work, nanoscale zero-valent iron (Fe0) supported on activated carbon (nZVI) was employed for eliminating Sb(V) from the drinking water. To better understand the overall process, the effects of several experimental variables, including pH, dissolved oxygen (DO), coexisting ions, and adsorption kinetics on the removal of Sb(V) from the SW were investigated by employing fixed-bed column runs or batch-adsorption methods. A pH of 4.5 and 72 h of equilibrium time were found to be the ideal conditions for drinking water. The presence of phosphate (PO43-), silicate (SiO42-), chromate (CrO42-) and arsenate (AsO43-) significantly decreased the rate of Sb(V) removal, while humic acid and other anions exhibited a negligible effect. The capacity for Sb(V) uptake decreased from 6.665 to 2.433 mg when the flow rate was increased from 5 to 10 mL·min−1. The dynamic adsorption penetration curves of Sb(V) were 116.4% and 144.1% with the weak magnetic field (WMF) in fixed-bed column runs. Considering the removal rate of Sb(V), reusability, operability, no release of Sb(V) after being incorporated into the iron (hydr)oxides structure, it can be concluded that WMF coupled with ZVI would be an effective Sb(V) immobilization technology for drinking water.

Mechanistic Approaches to the Light-Induced Neural Cell Differentiation: Photobiomodulation vs Low-Dose Photodynamic Therapy

Neurodegenerative diseases are the results of irreversible damages in the neuronal cells by affecting vital functions temporarily or even permanently. The use of light for the treatment of these diseases is an emerging promising innovative method. Photobiomodulation (PBM) and Photodynamic Therapy (PDT) are the modalities that have a wide range of use in medicine and have opposite purposes, biostimulation and cell death respectively. In this study, we aimed to compare these two modalities (PDT and PBM) at low-level intensities and create a stimulatory effect on the differentiation of PC12 cells. Three different energy densities (1, 3, and 5 J/cm 2 ) were used in PBM and Chlorin e6-mediated PDT applications upon irradiation with 655-nm laser light.The light-induced differentiation profile of PC12 cells was analyzed by morphological examinations, qRT-PCR, cell viability assay, and some mechanistic approaches such as; the analysis of intracellular ROS production, NO release, and mitochondrial membrane potential change. It has been observed that both of these modalities were successful at neural cell differentiation. PBM at 1 J/cm 2 and low-dose PDT at 3 J/cm 2 energy densities provided the best differentiation profiles which were proved by the over-expressions of SYN-1 and GAP43 genes. It was also observed that intracellular ROS production and NO release had pivotal roles in these mechanisms with more cell differentiation obtained especially in low-dose PDT application. It can be concluded that light-induced mechanisms with properly optimized light parameters have the capacity for neural cell regeneration and thus, can be a successful treatment for incurable neurodegenerative diseases.

Abstract P250: A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro , which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro . Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM (5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 ± 12.09%), Ang II (+28.76 ± 17.65%) and Ang-(1-5) (+78.00 ± 23.82%) increased NO release (one-way ANOVA, p < 0.05 vs control, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ± 4.74%). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 ±10.24%; Ang II -4.79 ± 12.44%; Ang (1-5) -8.88 ± 18.16%; Ang-(1-7) -11.57 ± 19.95%) indicating that the responses in CHO-AT2 were AT2R specific. Conclusion: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence in an automated way is suitable as high-throughput assay for the identification of AT2R-agonistic compounds in vitro , Application of the assay revealed that Ang-(1-5), which is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

Evaluation of Leishmania infantum 30x biotherapy effects in the prevention and treatment of visceral leishmaniasis: in vivo and in vitro studies

Background: Leishmaniasis is a serious public health problem especially in developing countries [1]. The therapeutic potential of biotherapics against several microorganism has been described in vitro [2,3] and in vivo studies [4,5,6,7,8,9]. Considering the resistance of leishmaniasis to conventional treatment as well as previous studies with biotherapic, we evaluated the effects of Leishmania infantum 30x (BioLi30x) biotherapy. Aim: evaluate the antileishmanial effects of BioLi30x in in vivo and in vitro models. Methodology: The in vivo experiments were performed using BALB/c mice (n=138), divided into 8 groups: G1-healthy, G2-infected with L. infantum, G3-BioLi30x pre-treated, G4-BioLi30x pre/post-treated, G5-BioLi30x post-treated, G6-H2O30x post-treated, G7-Antimonium crudum 30x post-treated and G8-Glucantime® post-treated. After 49 days of treatment, the animals were submitted to euthanasia (ethical approval ECUA/UFRJ/066/14). Liver and spleen histological changes were evaluated, and serum samples were aliquoted and storage at -20°C for cytokine assays. The in vitro assays were performed using RAW 264.7 macrophages treated with BioLi30x and infected with L. infantum. The morphological aspects were evaluated by scanning electron microscopy (SEM), and the nitric oxide (NO) release was quantified in the supernatant of infected macrophages. Results: The histological analysis from 4 independent experiments showed livers with normal appearance (G1); periportal chronic hepatitis (G2,G4,G5,G8); discreet (G3,G7), moderate (G4,G5,G6), and severe (G2,G8) vacuolar hydropic degeneration; congestion and neutrophilic inflammation (G2,G4,G5,G6,G8), and possible amastigotes within macrophages (G2-G8). Spleens presented healthy appearance only in G1. All treated animals presented histological alterations, with different lesions severity, which involved spleen pulp hyperplasia with moderate disruption (G2,G8), as well as megakaryocytes and macrophages proliferation (G2- G8). SEM analyses showed BioLi30x treatments induced significant protozoan morphology alterations when compared to H2O30x. Besides, a 19% increase in the NO release was detected in RAW supernatants, when compared to H2O30x. Conclusions: BioLi30x and Antimonium crudum 30x modified the infection animal process, involving several cellular mechanisms as well as different histological damage. The in vitro experiments will be repeated in order to confirm these preliminary results.