Novel gramine-based bioconjugates obtained by click chemistry as cytoprotective compounds and potent antibacterial and antifungal agents.

A series of indole-1,4-disubstituted-1,2,3-triazole conjugates were synthesised by click chemistry. The haemolytic properties and cytoprotective activity of all the newly synthesised indole-triazole conjugates were tested in vitro. In addition, molecular docking was performed in silico for the selected conjugates to determine their antibacterial and antifungal properties. The results indicate that indole-triazole derivatives effectively protect human erythrocytes against free radical-induced haemolysis in a structure-dependent manner and that bis-indole-bis-triazole derivatives with alkyl linkers are excellent cytoprotective agents against oxidative haemolysis. The tested series of indole-1,4-disubstituted-1,2,3-triazole conjugates may have an affinity for the active sites of specific protein domains (PDB IDs: 2Q85 and 5V5Z) according to molecular docking studies.

Click chemistry as a method for the synthesis of steroid bioconjugates of bile acids derivatives and sterols.

Six steroid conjugates of bile acids and sterol derivatives have been synthesized using the click chemistry method. The azide-alkyne Huisgen cycloaddition of the propionyl ester of lithocholic, deoxycholic and cholic acid with azide derivatives of cholesterol and cholestanol gave new bile acid-sterol conjugates linked with a 1,2,3-triazole ring. Previously, sterols were converted to bromoacetate substituted derivatives by reaction with bromoacetic acid bromide in anhydrous dichloromethane. These compounds were then converted to azide derivatives using sodium azide. The propiolic esters of lithocholic, deoxycholic and cholic acids were obtained by reaction with propiolic acid in the presence of p-toluenesulfonic acid. Additionally, two of these steroids: methyl 3α-propynoyloxy-12α-acetoxy-5ß-cholane-24-oate and methyl 3α-propynoyloxy-7 α,12α-diacetoxy-5ß-cholane-24-oate were also obtained and characterized for the first time. All conjugates were obtained in good yields using an efficient synthesis method. The structures of all conjugates and the four substrates were confirmed by spectral (1H- and 13C NMR, FT-IR) analysis, mass spectrometry (ESI-MS), and PM5 semiempirical methods. The pharmacotherapeutic potential of the synthesized compounds was estimated based on the in silico Prediction of Activity Spectra for Substances (PASS) method. The cytotoxicity of the compounds was in vitro evaluated in a hemolytic assay using human erythrocytes as a cell model. The in silico and in vitro study results indicate that the selected compound possesses an interesting biological activity and can be considered as potential drug design agent. Additionally, molecular docking was performed for the selected conjugate.

Synthesis, Structure and Biological Activity of Indole-Imidazole Complexes with ZnCl2: Can Coordination Enhance the Functionality of Bioactive Ligands?

The ability of the indole-imidazole hybrid ligands to coordinate with the Zn(II) ion and the resulting structures of this new class of coordination compounds were analyzed in order to determine their structural properties and biological functionalities. For this purpose, six novel Zn(II) complexes, [Zn(InIm)2Cl2] (1), [Zn(InMeIm)2Cl2] (2), [Zn(IniPrIm)2Cl2] (3), [Zn(InEtMeIm)2Cl2] (4), [Zn(InPhIm)2Cl2] (5) and [Zn2(InBzIm)2Cl2] (6) (where InIm is 3-((1H-imidazol-1-yl)methyl)-1H-indole), were synthesized by the reactions of ZnCl2 and the corresponding ligand in a 1:2 molar ratio in methanol solvent at an ambient temperature. The structural and spectral characterization of these complexes was performed using NMR, FT-IR and ESI-MS spectrometry and elemental analysis, and the crystal structures of 1-5 were determined using single-crystal X-ray diffraction. Complexes 1-5 form polar supramolecular aggregates by utilizing, for this purpose, the N-H(indole)∙∙∙Cl(chloride) intermolecular hydrogen bonds. The assemblies thus formed differ depending on the distinctive molecular shape, which can be either compact or extended. All complexes were screened for their hemolytic, cytoprotective, antifungal, and antibacterial activities. The results show that the cytoprotective activity of the indole/imidazole ligand significantly increases upon its complexation with ZnCl2 up to a value comparable with the standard antioxidant Trolox, while the response of its substituted analogues is diverse and less pronounced.

Direct Visualization of the Dynamic Process of Epsilon Toxin on Hemolysis.

Hemolysis is the process of rupturing erythrocytes (red blood cells) by forming nanopores on their membranes using hemolysins, which then impede membrane permeability. However, the self-assembly process before the state of transmembrane pores and underlying mechanisms of conformational change are not fully understood. In this work, theoretical and experimental evidence of the pre-pore morphology of Clostridium perfringens epsilon toxin (ETX), a typical hemolysin, is provided using in situ atomic force microscopy (AFM) complemented by molecular dynamics (MD) simulations to detect the conformational distribution of different states in Mica. The AFM suggests that the ETX pore is formed in two stages: ETX monomers first attach to the membrane and form a pre-pore in no special conditions required, which then undergo a conformational change to form a transmembrane pore at temperatures above the critical point in the presence of receptors. The authors' MD simulations reveal that initial nucleation occurs when specific amino acids adsorb to negatively charged mica cavities. This work fills the knowledge gap in understanding the early stage of hemolysis and the oligomerization of hemolysins. Moreover, the newly identified pre-pore of ETX holds promise as a candidate for nanopore applications.

A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens.

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.

Protective effect of rutin against thiram-induced cytotoxicity and oxidative damage in human erythrocytes.

Thiram is a fungicide that is used to prevent fungal diseases in seeds and crops and also as an animal repellent. The pro-oxidant activity of thiram is well established. Rutin is a flavonoid glycoside present in many fruits and plants and has several beneficial properties, including antioxidant effects. We have previously shown that thiram causes oxidative damage in human erythrocytes. The present study was designed to evaluate the protective effect of rutin against thiram-induced damage in human erythrocytes. Treatment of erythrocytes with 0.5 mM thiram for 4 h increased the level of oxidative stress markers, decreased antioxidant power and lowered the activity of antioxidant and membrane bound enzymes. It also enhanced the generation of reactive oxygen and nitrogen species (ROS and RNS) and altered the morphology of erythrocytes. However, prior treatment of erythrocytes with rutin (0.5, 1 and 2 mM) for 2 h, followed by 4 h incubation with 0.5 mM thiram, led to a decrease in the level of oxidative stress markers in a rutin concentration-dependent manner. A significant restoration in the antioxidant power and activity of antioxidant enzymes was observed upon the treatment of erythrocytes with 1 and 2 mM rutin. Pre-incubation with rutin lowered the generation of ROS and RNS which will reduce oxidative damage in erythrocytes. The thiram-induced changes in cell morphology and activity of membrane-bound enzymes were also attenuated by rutin. These results suggest that rutin can be used to mitigate thiram-induced oxidative damage in human erythrocytes.

Sulfhemoglobin under the spotlight - Detection and characterization of SHb and HbFeIII-SH.

Sulfhemoglobinemia is an incurable disease caused by an overdose of sulfur-containing drugs with oxidizing properties. Its diagnosis remains hindered due to the similarity of symptoms to other pathological state - methemoglobinemia, as well as contradictory information on the structure and characteristics of sulfhemoglobin. Herein, we present sulfhemoglobinemia model on living functional human erythrocytes, designed to recreate processes which could take place in a patient body in order to complement missing information and highlight distinctiveness of two hemoglobin (Hb) adducts formed after interaction with sulfur donors. Employed techniques, UV-Vis absorption, Raman, Fourier transformed infrared (FT-IR) and electronic circular dichroism (ECD) spectroscopies, allowed to distinguish and characterize Hb adduct with sulfur atom bounded directly to the iron ion (HbFeIII-SH), and irreversibly connected to the porphyrin ring (SHb - sulfhemoglobin). Presented herein results provided also new evidence on formation of both these hemoglobin adducts inside functional erythrocytes under oxidative conditions and during sulfur-containing drug presence, what can be further translated into future physiological studies. Moreover, we found that sulfur attachment to the porphyrin ring altered Hb structure and lead to changes in protein packing inside RBCs, eventually. Interestingly, measurement of blood drop smear by Raman spectroscopy occurred the most accurate method to differentiate HbFeIII-SH and SHb, indicating potential of this technique in sulfhemoglobinemia diagnosis.

Eritrocitos humanos como biomodelo farmacológico de citoprotección antioxidante para la evaluación de nuevas alternativas terapéuticas en pacientes con COVID-19 / Human erythrocytes as pharmacological biomodel of antioxidant cytoprotection for the evaluation of new therapeutic alternatives in patients with COVID-19

Introducción: El estrés oxidativo puede afectar las membranas biológicas de diferentes tipos celulares en el organismo, lo cual se ha evidenciado en los daños a los tejidos y órganos de pacientes con COVID-19, por lo cual las investigaciones recientes están relacionadas con la búsqueda de fármacos citoprotectores y antioxidantes que minimicen estos daños. Objetivo: Evaluar los eritrocitos humanos como biomodelo farmacológico de citoprotección antioxidante. Métodos: Se evaluó el modelo de citotoxicidad en eritrocitos inducido por peróxido de hidrógeno y se valoró el sistema de diagnóstico propuesto en un ensayo de citoprotección en eritrocitos, con el empleo del ácido ascórbico como sustancia de referencia. Resultados: Para la concentración de eritrocitos utilizada se logró un modelo de citotoxicidad a la concentración de 10 mM de peróxido a los 30 minutos de incubación. La sustancia de referencia empleada no mostró signos de citotoxicidad en el test de hemólisis. En el ensayo de citoprotección se evidenció un efecto farmacológico del referente, con un valor del índice de citoprotección de 12,71 µg/mL. El estudio de microscopía óptica mostró daños morfológicos severos en los eritrocitos tratados con peróxido de tipo esferocitos, equinocitos y esferoequinocitos, que disminuyeron significativamente en presencia de dicha sustancia de referencia. Conclusiones: El biomodelo farmacológico propuesto puede ser empleado en la evaluación de nuevas alternativas terapéuticas con propiedades citoprotectoras antioxidantes para el tratamiento de pacientes con COVID-19.

Introduction: The oxidative stress can affect the biological membranes of different cellular types in the organism, which has been evidenced in the damages to the tissues and organs of patients with COVID-19, reason why the recent investigations are related to the search of cytoprotector and antioxidant drugs that minimize these damages. Objective: To evaluate the human erythrocytes as pharmacological biomodel of antioxidant cytoprotection. Methods: The cytotoxicity pattern was evaluated in erythrocytes induced by peroxide of hydrogen and the system of diagnosis proposed was valued in a cytoprotection assay in erythrocytes, with the use of ascorbic acid as reference substance. Results: For the concentration of erythrocytes used a cytotoxicity model was achieved to the concentration of 10 mM of peroxide at 30 minutes of incubation. The substance of reference used didn't show cytotoxicity signs in the hemolysis test. In the cytoprotection assay a pharmacological effect of the referent was evidenced, with a value of the cytoprotection index of 12.71 µg/mL. The study of optic microscopy showed severe morphological damages in the erythrocytes treated with peroxide of spherocytes, echinocytes and spheroechinocytes type that significantly diminished in presence of this reference substance. Conclusions: The proposed pharmacological biomodel can be used in the evaluation of new therapeutic alternatives with antioxidant cytoprotector properties for the treatment of patients with COVID-19.

Direct Attachment with Erythrocytes Augments Extracellular Growth of Pathogenic Mycobacteria.

Pathogenic intracellular mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium avium, which cause lung diseases, can grow in macrophages. Extracellular mycobacteria have been reported in the lungs, blood, and sputum of patients, indicating the involvement of these pathogens in disease progression. Erythrocytes are involved in the symptoms associated with pulmonary mycobacterial diseases, such as bloody sputum and hemoptysis; however, little attention has been paid to the role of erythrocytes in mycobacterial diseases. Herein, we found that Mycobacterium avium subsp. hominissuis (MAH) and Mycobacterium intracellulare colocalized with erythrocytes at the sites of lung infection, inside capillaries and necrotic areas of granulomas, using histopathological examinations. Electron microscopy showed that MAH adhered and entered human erythrocytes when they were cocultured in vitro. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins. Importantly, MAH grew vigorously without causing any pronounced damage to erythrocytes. This erythrocyte-mediated enhancement of MAH growth occurred extracellularly depending on its direct attachment to erythrocytes. In contrast, MAH failed to multiply inside erythrocytes. Similarly, erythrocytes augmented the growth of other pathogenic mycobacteria, such as M. intracellulare and M. tuberculosis. THP-1 cell-derived human macrophages preferentially phagocytosed erythrocytes that were attached to mycobacteria (compared to bacteria alone), suggesting that erythrocyte-attached mycobacteria are an efficient infectious source for macrophages. Our findings provide new insights into the pathogenesis of mycobacterial diseases and offer an alternative and useful strategy for treating mycobacterial disease. IMPORTANCE Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare, cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites. Subsequent studies demonstrated that direct interaction with erythrocytes enhances the extracellular proliferation of mycobacteria based on the following results: 1. MAH adhered and invaded human erythrocytes upon coculture in vitro; 2. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins; 3. MAH rapidly proliferated when directly attached to erythrocytes but not within them; 4. other mycobacteria, such as M. intracellulare and M. tuberculosis, also proliferated in the same way as MAH. The finding that pathogenic mycobacteria grow extracellularly in an erythrocyte-dependent manner is of considerable clinical importance for understanding disease progression and latent infection.

New triazole-bearing gramine derivatives - synthesis, structural analysis and protective effect against oxidative haemolysis.

The new series of triazole-bearing gramine derivatives were synthesized through a CuAAC procedure. The structures of all newly obtained compounds were confirmed by spectroscopic analysis and DFT methods. The obtained derivatives were screened for their protective potency against oxidative haemolysis induced by free radicals generated from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). Our work demonstrates that derivatives with propyl or octyl linker and phthalimide group associated with indole-triazole moiety, which have a folded structure, effectively protect human erythrocytes against oxidative stress-induced haemolysis.

Chemical composition of Gastrocotyle hispida (Forssk.) bunge and Heliotropium crispum Desf. and evaluation of their multiple in vitro biological potentials.

Medicinal plants largely serve as a source of bioactive compounds in traditional medicines to cure various diseases. The present study was aimed at chemical composition, antioxidant, antimicrobial, cytotoxic and antihemolytic potential of five different extracts of G. hispida and H. crispum (Boraginaceae). G. hispida methanolic extract displayed highest number (eleven) of polyphenolic compounds by using high performance liquid chromatography (HPLC). Functional groups were identified by Fourier-transformed infrared spectroscopy (FTIR) and elements (Si, Fe, Ba, Mg, Ti, Ca, Mg and Cr) were observed by using laser-induced breakdown spectroscopy (LIBS) which were also highly expressed in G. hispida as compared to H. crispum. Antioxidant activity was determined via six assays and antibacterial activity was observed in decreasing order of methanol > ethanol > chloroform > ethyl acetate > n-Hexane in both species. Cytotoxic potential was investigated against brine shrimps and then liver (HepG2) and skin (HT144) cancer cell lines which was detected highest in the G. hispida ethanolic extract (50.76 % and 72.95 %). However, H. crispum chloroform extract revealed highest (31.869 µg/mL) antihemolytic activity and its methanolic extract indicated highest (13.5 %) alpha-amylase inhibitory potential. Altogether, results suggested that both species could be used effectively in food and drug industries owing to the presence of vital bioactive compounds and elements. In future, we recommend to isolate active compounds and to perform in vivo biological assays to further validate their potential biological applications.

Human erythrocytes, nuclear factor kappaB (NFκB) and hydrogen sulfide (H2S) - from non-genomic to genomic research.

Enucleated mature human erythrocytes possess NFĸBs and their upstream kinases. There is a negative correlation between eryptosis (cell death of erythrocytes) and the amount of NFĸB subunits p50 and Rel A (p65). This finding is based on the fact that young erythrocytes have the highest levels of NFĸBs and the lowest eryptosis rate, while in old erythrocytes the opposite ratio prevails. Human erythrocytes (hRBCs) effectively control the homeostasis of the cell membrane permeable anti-inflammatory signal molecule hydrogen sulfide (H2S). They endogenously produce H2S via both non-enzymic (glutathione-dependent) and enzymic processes (mercaptopyruvate sulfur transferase-dependent). They uptake H2S from diverse tissues and very effectively degrade H2S via methemoglobin (Hb-Fe3+)-catalyzed oxidation. Interestingly, a reciprocal correlation exists between the intensity of inflammatory diseases and endogenous levels of H2S. H2S deficiency has been observed in patients with diabetes, psoriasis, obesity, and chronic kidney disease (CKD). Furthermore, endogenous H2S deficiency results in impaired renal erythropoietin (EPO) production and EPO-dependent erythropoiesis. In general we can say: dynamic reciprocal interaction between tumor suppressor and oncoproteins, orchestrated and sequential activation of pro-inflammatory NFĸB heterodimers (RelA-p50) and the anti-inflammatory NFĸB-p50 homodimers for optimal inflammation response, appropriate generation, subsequent degradation of H2S etc., are prerequisites for a functioning cell and organism. Diseases arise when the fragile balance between different signaling pathways that keep each other in check is permanently disturbed. This work deals with the intact anti-inflammatory hRBCs and their role as guarantors to maintain the redox status in the physiological range, a basis for general health and well-being.

Changes in Human Erythrocyte Exposed to Organophosphate Flame Retardants: Tris(2-chloroethyl) Phosphate and Tris(1-chloro-2-propyl) Phosphate.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are the main representatives of organophosphate flame retardants (OPFRs). The exposure of humans to OPFRs present in air, water, and food leads to their occurrence in the circulation. Thus far, no report has been published about the influence of these retardants on non-nucleated cells like mature erythrocytes. Therefore, the impact of TCEP and TCPP (in concentrations determined in human blood as well as potentially present in the human body after intoxication) on human erythrocytes was evaluated. In this study, the effect of TCEP and TCPP on the levels of methemoglobin, reduced glutathione (GHS), and reactive oxygen species (ROS), as well as the activity of antioxidative enzymes, was assessed. Moreover, morphological, hemolytic, and apoptotic alterations in red blood cells were examined. Erythrocytes were incubated for 24 h with retardants in concentrations ranging from 0.001 to 1000 µg/mL. This study has revealed that the tested flame retardants only in very high concentrations disturbed redox balance; increased ROS and methemoglobin levels; and induced morphological changes, hemolysis, and eryptosis in the studied cells. The tested compounds have not changed the activity of the antioxidative system in erythrocytes. TCPP exhibited a stronger oxidative, eryptotic, and hemolytic potential than TCEP in human red blood cells. Comparison of these findings with hitherto published data confirms a much lower toxicity of OPFRs in comparison with brominated flame retardants.

Wild garlic extract reduces lipid peroxidation in terbuthylazine-treated human erythrocytes.

Background: Among other negative effects, herbicides induce oxidative stress, leading to lipid peroxidation and protein oxidation. Therefore, there is a growing need to identify natural compounds with sufficient antioxidant capacity and mitigate the negative effects of herbicides without side effects.Objective: Our study aimed to examine the protective effect of the phenolic extract of wild garlic (WG) leaves on terbuthylazine-treated erythrocytes.Material and methods: In human erythrocytes treated with the herbicide terbuthylazine (4.5 mg/L) alone and a combination of terbuthylazine and WG extract, we measured malondialdehyde (MDA) and haemoglobin (Hb) concentrations and the antioxidant activities of CuZn superoxide dismutase (SOD1; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) in vitro.Results: In comparison with terbuthylazine, WG extract reduced the concentrations of MDA and Hb from 59.69 to 43.45 nmol/gHb (27%, p < 0.001) and 165.08 to 128.64 g/L (22%, p < 0.05), respectively. Catalase activity was induced for samples treated with both WG extract and terbuthylazine compared with terbuthylazine alone (p < 0.05).Conclusions: The results demonstrated that WG may reduce the toxicity of terbuthylazine, and the erythrocyte membrane may be the primary site of phenolic action. Therefore, the lipid peroxidation intensity could be a biomarker of oxidative damage caused by terbuthylazine and the protective effect of WG.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Ligand-Sensitised LaF3 :Eu3+ and SrF2 :Eu3+ Nanoparticles and in Vitro Haemocompatiblity Studies.

Luminescent Ln3+ -doped nanoparticles (NPs) functionalised with the desired organic ligand molecules for haemocompatibility studies were obtained in a one-pot synthesis. Chelated aromatic organic ligands such as isophthalic acid, terephthalic acid, ibuprofen, aspirin, 1,2,4,5-benzenetetracarboxylic acid, 2,6-pyridine dicarboxylic acid and adenosine were applied for surface functionalisation. The modification of the nanoparticles is based on the donor-acceptor character of the ligand-nanoparticle system, which is an alternative to covalent functionalisation by peptide bonding as presented in our recent report. The aromatic groups of selected ligands absorb UV light and transfer their excited-state energy to the dopant Eu3+ ions in LaF3 and SrF2 NPs. Herein, we discuss the structural and spectroscopic characterisation of the NPs and the results of haemocompatibility studies. Flow cytometry analysis of the nanoparticles' membrane-binding is also presented.

Synthesis and Characterization of Mesoporous Mg- and Sr-Doped Nanoparticles for Moxifloxacin Drug Delivery in Promising Tissue Engineering Applications.

Mesoporous silica-based nanoparticles (MSNs) are considered promising drug carriers because of their ordered pore structure, which permits high drug loading and release capacity. The dissolution of Si and Ca from MSNs can trigger osteogenic differentiation of stem cells towards extracellular matrix calcification, while Mg and Sr constitute key elements of bone biology and metabolism. The aim of this study was the synthesis and characterization of sol-gel-derived MSNs co-doped with Ca, Mg and Sr. Their physico-chemical properties were investigated by X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), Brunauer Emmett Teller and Brunauer Joyner Halenda (BET/BJH), dynamic light scattering (DLS) and ζ-potential measurements. Moxifloxacin loading and release profiles were assessed with high performance liquid chromatography (HPLC) cell viability on human periodontal ligament fibroblasts and their hemolytic activity in contact with human red blood cells (RBCs) at various concentrations were also investigated. Doped MSNs generally retained their textural characteristics, while different compositions affected particle size, hemolytic activity and moxifloxacin loading/release profiles. All co-doped MSNs revealed the formation of hydroxycarbonate apatite on their surface after immersion in simulated body fluid (SBF) and promoted mitochondrial activity and cell proliferation.

A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity.

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

Polysaccharide from Lycium arabicum: Structural Features, in Vitro Antioxidant Activities and Protective Effect against Oxidative Damage in Human Erythrocytes.

In this research work, a water-soluble polysaccharide (LAP) isolated from the fruits of Lycium arabicum was investigated. LAP contains carbohydrates (82.45±1.23 %), protein (1.56±0.21 %), and uronic acids (3.56±0.34 %). The analysis of the monosaccharide composition revealed the presence of rhamnose, arabinose, galactose, glucose and mannose in a molar ratio of 4.7 : 1.5 : 1 : 8.7 : 16.4 : 5.6. The extracted polysaccharide (PS) was considered as heterogeneous and highly branched by interpreting its GC/MS, FT-IR and NMR data. Crystallinity of LAP was inferred from its X-ray diffractometry (XRD) and Scanning Electron Microscopy (SEM) analysis. LAP exhibited an interesting stability at high temperatures (∼254 °C) and in a wide range of pH (3-9) deduced, respectively, from its DSC and zeta potential analysis. LAP displayed a strong antioxidant activity at low concentrations evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging, ferric reducing activity power (FRAP), free radical scavenging ability, superoxide radical-scavenging and hydroxyl radical-scavenging abilities. Inhibition of erythrocyte hemolysis and lipid peroxidation was also assessed. In 5 h, LAP treatment allowed the protection of the damaged erythrocytes caused by AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), to reduce the level of malondialdehyde (MDA) as well as to increase the reduced glutathione (GSH) level.

Effect of the Solvent on Propolis Phenolic Profile and its Antifungal, Antioxidant, and In Vitro Cytoprotective Activity in Human Erythrocytes Under Oxidative Stress.

Propolis is a natural bee product with various beneficial biological effects. The health-promoting properties of propolis depend on its chemical composition, particularly the presence of phenolic compounds. The aim of this study was to evaluate the relationship between extraction solvent (acetone 100%, ethanol 70% and 96%) and the antifungal, antioxidant, and cytoprotective activity of the extracts obtained from propolis. Concentrations of flavonoids and phenolic acids in the propolis extracts were determined using ultrahigh-performance liquid chromatography. The antioxidant potential of different extracts was assessed on the basis of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free-radical-scavenging activity, Fe3+-reducing power, and ferrous ion (Fe2+)-chelating activity assays. The ability of the extracts to protect human red blood cell membranes against free-radical-induced damage and their antifungal activity was also determined. The results showed that the concentration of flavonoids in the propolis extracts was dependent on the solvent used in the extraction process and pinocembrin, chrysin, galangin, and coumaric acid were the most abundant phenols. All extracts exhibited high antioxidant potential and significantly protected human erythrocytes against oxidative damage. On the other hand, the antifungal activity of the propolis extracts depended on the solvent used in extraction and the fungal strains tested. It needs to be stressed that, to the best of our knowledge, there is no study relating the effect of solvent used for extraction of Polish propolis to its phenolic profile, and its antifungal, antioxidant, and cytoprotective activity.