Structural investigation of interactions between halogenated flavonoids and the lipid membrane along with their role as cytotoxic agents.

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use.

Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.

[Immunogenic and toxic effects of graphene oxide nanoparticles in mouse skeletal muscles and human red blood cells].

OBJECTIVE: To investigate immunogenic and toxic effects of graphene oxide (GO) nanoparticles in mouse skeletal muscles and in human blood in vitro. METHODS: GO nanoparticles prepared using a probe sonicator were supended in deionized H2O or PBS, and particle size and surface charge of the nanoparticles were measured with dynamic light scattering (DLS). Different concentrations (0.5, 1.0 and 2.0 mg/mL) of GO suspension or PBS were injected at multiple sites in the gastrocnemius muscle (GN) of C57BL/6 mice, and inflammatory response and immune cell infiltrations were detected with HE and immunofluorescence staining. We also examined the effects of GO nanoparticles on human red blood cell (RBC) morphology, hemolysis and blood coagulation using scanning electron microscope (SEM), spectrophotometry, and thromboelastography (TEG). RESULTS: GO nanoparticles suspended in PBS exhibited better colloidal dispersity, stability and surface charge effects than those in deionized H2O. In mouse GNs, injection of GO suspensions dose- and time-dependently resulted in sustained muscular inflammation and myofiber degeneration at the injection sites, which lasted till 8 weeks after the injection; immunofluorescence staining revealed obvious infiltration of monocytes, macrophages, dendritic cells and CD4+ T cells around the injection sites in mouse GNs. In human RBCs, incubation with GO suspensions at 0.2, 2.0 and 20 mg/mL, but not at 0.002 or 0.02 mg/mL, caused significant alterations of cell morphology and hemolysis. TEG analysis showed significant abnormalities of blood coagulation parameters following treatment with high concentrations of GO. CONCLUSION: GO nanoparticles can induce sustained inflammatory and immunological responses in mouse GNs and cause RBC hemolysis and blood coagulation impairment, suggesting its muscular toxicity and hematotoxicity at high concentrations.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

In vivo determination of the anti-inflammatory and antioxidant effects of the aqueous extract of Syzygium aromaticum (clove) in an asthmatic rat model.

Asthma is a chronic inflammatory disease of the airways strongly associated with interleukin-4 (IL-4), a cytokine that mediates and regulates various immune responses, including allergic reactions. This study aimed to evaluate the anti-inflammatory and antioxidant effects of an Aqueous Extract of Clove (AEC) Syzygium aromaticum on the lungs and erythrocytes of an experimental asthma model in Wistar rats. For this purpose, four groups of male rats were examined: control, sensitized with ovalbumin (OVA), treated with AEC, and treated with a combination of OVA/AEC. After treatment, the antioxidant effect was determined by measuring the malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione (GSH), and catalase (CAT) levels. The anti-inflammatory effect was determined by measuring IL-4 levels by performing enzyme-linked immunosorbent assay (ELISA) using serum, lung, and bronchoalveolar lavage fluid (BALF) samples. A significant reduction (p ≤ 0.05) in the MDA levels and a significant increase (p ≤ 0.05) in the levels of GPx and CAT were observed in the lungs of rats treated with cloves. However, no statistically significant variation was observed in GSH levels. In erythrocytes, no statistically significant differences were observed between the experimental batches. Regarding the anti-inflammatory effect, the administration of S. aromaticum extract to sensitized rats resulted in a recovery in the levels of total proteins and IL-4 and a decrease in the three compartments studied (lungs, serum, and bronchoalveolar liquid). These results were confirmed by microscopic examination of lung histological sections. Overall, these findings confirmed that the AEC has anti-inflammatory and antioxidant effects.

Intelligent berberine-loaded erythrocytes attenuated inflammatory cytokine productions in macrophages.

Erythrocytes are impressive tools for drug delivery, especially to macrophages. Therefore, berberine was loaded into erythrocytes using both hypotonic pre-swelling and endocytosis methods to target macrophages. Physicochemical and kinetic parameters of the resulting carrier cells, such as drug loading/release kinetics, osmotic fragility, and hematological indices, were determined. Drug loading was optimized for the study using Taguchi experimental design and lab experiments. Loaded erythrocytes were targeted to macrophages using ZnCl2 and bis-sulfosuccinimidyl-suberate, and targeting was evaluated using flow cytometry and Wright-Giemsa staining. Differentiated macrophages were stimulated with lipopolysaccharide, and the inflammatory profiles of macrophages were evaluated using ELISA, western blotting, and real-time PCR. Findings indicated that the endocytosis method is preferred due to its low impact on the erythrocyte's structural integrity. Maximum loading achieved (1386.68 ± 22.43 µg/ml) at 1500 µg/ml berberine treatment at 37 °C for 2 h. Berberine successfully inhibited NF-κB translation in macrophages, and inflammatory response markers such as IL-1ß, IL-8, IL-23, and TNF-α were decreased by approximately ninefold, sixfold, twofold, eightfold, and twofold, respectively, compared to the LPS-treated macrophages. It was concluded that berberine-loaded erythrocytes can effectively target macrophages and modulate the inflammatory response.

Chitosan/starch based unoxidized tannic acid modified microparticles for rapid hemostasis with broad spectrum antibacterial activity.

The development of a rapid hemostat through a facile method with co-existing antibacterial activity and minimum erythrocyte lysis property stands as a major requirement in the field of hemostasis. Herein, a series of novel microparticle hemostats were synthesized using chitosan, different hydrothermally-treated starches, and cross-linked with tannic acid (TA) simultaneously in an unoxidized environment via ionotropic gelation method. Hemostats' comparative functional properties, such as adjustable antibacterial and erythrocyte compatibility upon various starch additions were evaluated. The in vivo hemostatic study revealed that the developed hemostats for mouse liver laceration and rat tail amputation had clotting times (13 s and 38 s, respectively) and blood loss (51 mg and 62 mg, respectively) similar to those of Celox™. The erythrocyte adhesion test suggested that erythrocyte distortion can be lowered by modifying the antibacterial hemostats with different starches. The broad-spectrum antibacterial efficacy of the hemostats remained intact against S. aureus (>90 %), E. coli (>80 %), and P. mirabilis bacteria upon starch modification. They also demonstrated high hemocompatibility (<3 % hemolysis ratio), moderate cell viability (>81 %), in vivo biodegradation, and angiogenesis indicating adequate biocompatibility and wound healing. The developed hemostats hold significant promise to be employed as rapid hemostatic agents for preventing major bleeding and bacterial infection in emergencies.

Carbon Monoxide Alleviates Post-ischemia-reperfusion Skeletal Muscle Injury and Systemic Inflammation.

Restoration of blood flow in skeletal muscle after a prolonged period of ischemia induces muscular ischemia-reperfusion injury, leading to local injury/dysfunction in muscles followed by systemic inflammatory responses. However, preventive/curative agents for skeletal muscle ischemia injury are unavailable in clinics to date. Increasing evidence has validated that carbon monoxide (CO) prevents the progression of ischemia-reperfusion injury in various organs owing to its versatile bioactivity. Previously, we developed a bioinspired CO donor, CO-bound red blood cells (CO-RBC), which mimics the dynamics of RBC-associated CO in the body. In the present study, we have tested the therapeutic potential of CO-RBC in muscular injury/dysfunction and secondary systemic inflammation induced by skeletal muscle ischemia-reperfusion. The results indicate that CO-RBC rather than RBC alone suppressed elevation of plasma creatine phosphokinase, a marker of muscular injury, in rats subjected to both hind limbs ischemia-reperfusion. In addition, the results of the treadmill walking test revealed a significantly decreased muscular motor function in RBC-treated rats subjected to both hind limbs ischemia-reperfusion than that in healthy rats, however, CO-RBC treatment facilitated sustained muscular motor functions after hind limbs ischemia-reperfusion. Furthermore, CO-RBC rather than RBC suppressed the production of tumour necrosis factor (TNF)-α and interleukin (IL)-6, which were upregulated by muscular ischemia-reperfusion. Interestingly, CO-RBC treatment induced higher levels of IL-10 compared to saline or RBC treatments. Based on these findings, we suggest that CO-RBC exhibits a suppressive effect against skeletal muscle injury/dysfunction and systemic inflammatory responses after skeletal muscle ischemia-reperfusion.

Antibacterial and Hemolytic Activity of Antimicrobial Hydrogels Utilizing Immobilized Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are viewed as potential compounds for the treatment of bacterial infections. Nevertheless, the successful translation of AMPs into clinical applications has been impeded primarily due to their low stability in biological environments and potential toxicological concerns at higher concentrations. The covalent attachment of AMPs to a material's surface has been sought to improve their stability. However, it is still an open question what is required to best perform such an attachment and the role of the support. In this work, six different AMPs were covalently attached to a long-ranged ordered amphiphilic hydrogel, with their antibacterial efficacy evaluated and compared to their performance when free in solution. Among the tested AMPs were four different versions of synthetic end-tagged AMPs where the sequence was altered to change the cationic residue as well as to vary the degree of hydrophobicity. Two previously well-studied AMPs, Piscidin 1 and Omiganan, were also included as comparisons. The antibacterial efficacy against Staphylococcus aureus remained largely consistent between free AMPs and those attached to surfaces. However, the activity pattern against Pseudomonas aeruginosa on hydrogel surfaces displayed a marked contrast to that observed in the solution. Additionally, all the AMPs showed varying degrees of hemolytic activity when in solution. This activity was entirely diminished, and all the AMPs were non-hemolytic when attached to the hydrogels.

Anti-inflammatory and anti-diabetic properties of indanone derivative isolated from Fernandoa adenophylla in vitro and in silico studies.

Fernandoa adenophylla, due to the presence of phytochemicals, has various beneficial properties and is used in folk medicine to treat many conditions. This study aimed to isolate indanone derivative from F. adenophylla root heartwood and assess in-vitro anti-inflammatory and anti-diabetic characteristics at varying concentrations. Heat-induced hemolysis and glucose uptake by yeast cells assays were conducted to evaluate these properties. Besides, docking analyses were performed on four molecular targets. These studies were combined with molecular dynamics simulations to elucidate the time-evolving inhibitory effect of selected inhibitors within the active pockets of the target proteins (COX-1 and COX-2). Indanone derivative (10-100 µM) inhibited the lysis of human red blood cells from 9.12 ± 0.75 to 72.82 ± 4.36% and, at 5-100 µM concentrations, it significantly increased the yeast cells' glucose uptake (5.16 ± 1.28% to 76.59 ± 1.62%). Concluding, the isolated indanone might act as an anti-diabetic agent by interacting with critical amino acid residues of 5' adenosine monophosphate-activated protein kinase (AMPK), and it showed a binding affinity with anti-inflammatory targets COX-1, COX-2, and TNF-α. Besides, the obtained results may help to consider the indanone derivative isolated from F. adenophylla as a promising candidate for drug delivery, subject to outcomes of further in vivo and clinical studies.

Eliminating extracellular autoinducing peptide signals inhibits the Staphylococcus aureus quorum sensing agr system.

An accessory gene regulator (agr) in the quorum sensing (QS) system in Staphylococcus aureus contributes to host infection, virulence factor production, and resistance to oxidative damage. Artificially maintaining the inactive state of agr QS impedes the host infection strategy of S. aureus and inhibits toxin production. The QS system performs intercellular signal transduction, which is activated by the mature autoinducer peptide (AIP). It is released from cells after AgrD peptide processing as an intercellular signal associated with increased bacterial cell density. This study evaluated the effectiveness of inhibiting agr QS wherein AIP trap carriers were made to coexist when culturing Staphylococcus aureus. Immersing a nitrocellulose (NC) membrane in Staphylococcus aureus ATCC 12600 culture inhibited QS-dependent α-hemolysin production, which significantly reduced the hemolysis ratio of sheep red blood cells by the culture supernatant. A quartz crystal microbalance analysis supported AIP adsorption onto the NC membrane. Adding the NC membrane during culture was found to maintain the expression levels of the agr QS gene agrA and α-hemolysin gene hla lower than that when it was not added. Eliminating extracellular AIP signals allowed agr QS to remain inactive and prevented QS-dependent α-hemolysin expression. Isolating intercellular signals secreted outside the cell is an effective strategy to suppress gene expression in bacterial cells that collaborate via intercellular signaling.

Supplementing Glucose Intake Reverses the Inflammation Induced by a High-Fat Diet by Increasing the Expression of Siglec-E Ligands on Erythrocytes.

Siglec-9/E is a cell surface receptor expressed on immune cells and can be activated by sialoglycan ligands to play an immunosuppressive role. Our previous study showed that increasing the expression of Siglec-9 (the human paralog of mouse Siglec-E) ligands maintains functionally quiescent immune cells in the bloodstream, but the biological effects of Siglec-9 ligand alteration on atherogenesis were not further explored. In the present study, we demonstrated that the atherosclerosis risk factor ox-LDL or a high-fat diet could decrease the expression of Siglec-9/E ligands on erythrocytes. Increased expression of Siglec-E ligands on erythrocytes caused by dietary supplementation with glucose (20% glucose) had anti-inflammatory effects, and the mechanism was associated with glucose intake. In high-fat diet-fed apoE-/- mice, glucose supplementation decreased the area of atherosclerotic lesions and peripheral inflammation. These data suggested that increased systemic inflammation is attenuated by increasing the expression of Siglec-9/E ligands on erythrocytes. Therefore, Siglec-9/E ligands might be valuable targets for atherosclerosis therapy.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

SAR443809: a selective inhibitor of the complement alternative pathway, targeting complement factor Bb.

Dysregulated activation of the complement system is implicated in the onset or progression of several diseases. Most clinical-stage complement inhibitors target the inactive complement proteins present at high concentrations in plasma, which increases target-mediated drug disposition and necessitates high drug levels to sustain therapeutic inhibition. Furthermore, many efforts are aimed at inhibiting only terminal pathway activity, which leaves opsonin-mediated effector functions intact. We describe the discovery of SAR443809, a specific inhibitor of the alternative pathway C3/C5 convertase (C3bBb). SAR443809 selectively binds to the activated form of factor B (factor Bb) and inhibits alternative pathway activity by blocking the cleavage of C3, leaving the initiation of classical and lectin complement pathways unaffected. Ex vivo experiments with patient-derived paroxysmal nocturnal hemoglobinuria erythrocytes show that, although terminal pathway inhibition via C5 blockade can effectively inhibit hemolysis, proximal complement inhibition with SAR443809 inhibits both hemolysis and C3b deposition, abrogating the propensity for extravascular hemolysis. Finally, intravenous and subcutaneous administration of the antibody in nonhuman primates demonstrated sustained inhibition of complement activity for several weeks after injection. Overall, SAR443809 shows strong potential for treatment of alternative pathway-mediated disorders.

Long-term storage protocol of reagent red blood cells treated with 0.01M dithiothreitol (DTT) for pre-transfusion testing of patients receiving anti-CD38 therapy, daratumumab.

OBJECTIVE: Use red blood cell stabilizer to store the antibody screening and antibody identification reagent red blood cells (RBCs) treated with 0.01 mol/L DTT and investigate its value in the pre-transfusion examinations of patients treated with daratumumab. METHOD: Determined the optimal incubation time for the 0.01 mol/L DTT-treated RBCs method by evaluating the effect of treatment at different time points. Added ID-CellStab to store DTT-treated RBCs, determined the maximum shelf life of reagent RBCs by monitoring the hemolysis index, and assessed changes in the antigenicity of blood group antigens on the surface of RBCs during storage with antibody reagents. RESULT: A protocol for long-term storage of reagent red blood cells treated with the 0.01 mol/L DTT method was established. The optimal incubation time was 40-50 min. RBCs could be stored stably for 18 days after adding ID-CellStab. The protocol was able to eliminate pan-agglutination caused by daratumumab, with no significant changes in the antigens of most blood group systems, except for some attenuation of K antigen and Duffy blood group system antigens during the storage period. CONCLUSION: The storage protocol of reagent RBCs based on the 0.01 mol/L DTT method does not affect the detection of most blood group antibodies and retains a certain degree of detection ability for anti-K antibodies, allowing patients treated with daratumumab to quickly perform pre-transfusion examinations, making up for the shortcomings of currently commercial reagent RBCs.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phase I/II/III clinical trials. Erythrocytes do not have a nucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin­V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Perfluorocarbons cause thrombocytopenia, changes in RBC morphology and death in a baboon model of systemic inflammation.

A perfluorocarbon (PFC) investigated for treatment of traumatic brain injury (TBI) delivers oxygen to support brain function, but causes transient thrombocytopenia. TBI can cause acute inflammation with resulting thrombocytopenia; an interaction between the PFC effects and TBI inflammation might exacerbate thrombocytopenia. Therefore, PFC effects on platelet (PLT) function and hemostasis in a lipopolysaccharide (LPS) model of inflammation in the baboon were studied. Animals were randomized to receive saline ±LPS, and ± one of two doses of PFC. PLT count, transmission electron microscopy, and microparticle populations were quantified at baseline (BL) and at 2, 24, 48, 72, and 96 hours; hemostatic parameters for aggregometry and for blood clotting were measured at baseline (BL) and days 3 and 4. Injection of vehicle and LPS caused thrombocytopenia within hours; PFCs caused delayed thrombocytopenia beginning 48 hours post-infusion. LPS+PFC produced a more prolonged PLT decline and decreased clot strength. LPS+PFC increased ADP-stimulated aggregation, but PFC alone did not. Microparticle abundance was greatest in the LPS+PFC groups. LPS+PFC caused diffuse microvascular hemorrhage and death in 2 of 5 baboons in the low dose LPS-PFC group and 2 of 2 in the high dose LPS-PFC group. Necropsy and histology suggested death was caused by shock associated with hemorrhage in multiple organs. Abnormal morphology of platelets and red blood cells were notable for PFC inclusions. In summary, PFC infusion caused clinically significant thrombocytopenia and exacerbated LPS-induced platelet activation. The interaction between these effects resulted in decreased hemostatic capacity, diffuse bleeding, shock and death.

Cadmium chloride generates cytotoxic reactive species that cause oxidative damage and morphological changes in human erythrocytes.

Cadmium chloride (CdCl2) is a widely used industrial compound that exhibits multiple organ toxicity. Cadmium is transported through blood where erythrocytes are exposed to its action. Here the effect of CdCl2 on human erythrocytes was examined under in vitro conditions. Human erythrocytes were treated with 0.01-0.5 mM CdCl2 for 24 h at 37 °C. Lysates were made from CdCl2 treated and untreated (control) cells and used for further analysis. CdCl2 treatment resulted in marked hemolysis of erythrocytes and oxidation of hemoglobin to methemoglobin. This will result in anemia and also reduce the oxygen carrying ability of erythrocytes. Hemoglobin oxidation was accompanied by degradation of heme and release of free ferrous iron moiety. Further analysis showed elevated lipid hydroperoxides and formation of advanced oxidation protein products along with reduction in total sulfhydryl content, indicating the generation of oxidative stress condition in the cell. Incubation of erythrocytes with CdCl2 enhanced generation of reactive oxygen and nitrogen species, decreased the antioxidant power and inhibited pathways of glucose metabolism. Plasma membrane was damaged as indicated by enhanced osmotic fragility and inhibition of membrane bound enzymes. This was confirmed by electron microscopy which showed formation of echinocytes. These results show that CdCl2 generates reactive species which impair the antioxidant system resulting in oxidative damage to erythrocytes.