Hemolysis Of Red Blood Cells Research Articles

Discovery of adjuvants with antibacterial potentiation activity against carbapenemase-producing Enterobacterales based on in silico virtual screening

Bacterial multi-drug resistance has become a concern worldwide, especially after the emergence of carbapenemases. Adjuvants with antibacterial potentiation activity can resensitise drug-resistant strains to carbapenems. However, only a few adjuvants with antibacterial potentiation activity are currently available in clinical practice. Here, we first docked the library containing more than 30,000 small molecules to carbapenemases including Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-5 (NDM-5), through in silico virtual screening to obtain lead compounds against carbapenemase-producing Enterobacterales. Meanwhile, the in vitro antibacterial potentiation assays revealed that ibandronate, azacytidine, ribostamycin sulfate and cidofovir exhibited synergistic or additive activity in the presence of meropenem, with good biocompatibility based on red blood cell hemolysis and cell viability tests. Furthermore, the combination of meropenem and azacytidine showed high efficacy in a mouse sepsis model infected with an NDM-5-producing clinical strain, with a 100% survival rate, decreased bacterial burden and alleviated pathological deterioration. These results suggest that the virtual screening is a promising strategy to identify new antibiotic adjuvants targeting carbapenemase-producing Enterobacterales.

Quality Parameters of Polyherbal Formulation of Three Different Marketed Brands of Vilwadi Gutika: A Comparative Evaluation Study

Background: Ayurveda is a highly beneficial medical system due to its natural components that effectively eliminate disease causes, restore balance, and prevent further recurrence. Polyherbal formulations are highly popular due to their wide therapeutic range, making them safe at high doses and producing minimal negative effects when taken improperly. The goal of this study was to standardize the efficacy and quality of commercial herbal tablet formulations. Methods: Three marketed samples of vilwadi Gutika ware used for the study. After authentication of the samples, the phytochemical stander like determination of pH, ash value, acid insoluble ash, water soluble ash, water soluble extractive, alcohol and soluble extractive were determined. In vitro anti-microbial activity was determined using Agar well diffusion method and the anti – inflammatory activity of Vilwadi tablet of different ayurvedic agency was assessed by in- vitro HRBC (Human Red Blood Cells) membrane stabilization method. Results: The physicochemical properties of polyherbal pills met Pharmacopeial criteria, including pH, ash value, hardness, and friability. Sample 1 was the most acceptable, with a noticeable hardness (0.93) for rapid disintegration. The sample 3 (Vilwadi gutika – Arya Vaidya Pharmacy, Coimbatore) exhibited the highest antimicrobial activity against E. coli, Staphylococcus sp, and Candida sp, while sample 1 showed the highest activity against E. coli and Staphylococcus sp, and less activity against Candida sp. The in vitro anti-inflammatory activity shown that at concentration of 100µg / ml, the test compound inhibited 66.6 % of RBC haemolysis as compared with 54.16 % produced by Indomethacin (positive control) at 100µg / ml. Conclusion: The investigation reveals that the Viladi gutika herbal formulation can be standardized using a scientific quality control technique, meeting Indian pharmacopoeia requirements for tablet dosage form, with further research needed to analyze other characteristics. Keywords: Viladi gutika, anti-microbial activity, anti-inflammatory activity.

Phytochemical and Biological Investigations of Bacopa monnieri L.

Bacopa monnieri L. (Family: Scrophulariaceae), a medicinal herb of Bangladesh, has been used in traditional medicine to increase memory and brain power. The present research was aimed to investigate the phytochemical composition and biological activities of whole plant of B. monnieri. Through phytochemical analysis, two triterpenes, taraxerone and betulinic acid were obtained from B. monnieri. The crude methanol extract of the whole plant of Bacopa monnieri and its petroleum ether, dichloromethane, chloroform and aqueous fractions were screened for determination of several in vitro biological activities. In DPPH assay for antioxidant test, the dichloromethane fraction showed strong DPPH radical quenching activity (IC50 = 3.78 μg/ml). The chloroform fraction exhibited the maximum lethal concentration, LC50 value of 5.5 μg/ml against brine shrimp. In membrane stabilization assay, the crude extract and all solvent fractions revealed significant anti-inflammatory activity by inhibiting hemolysis of RBC induced by both hypotonic solution and heat. In thrombolytic assay, the petroleum ether fraction demonstrated highest thrombolytic activity (39.24%), when compared to the standard streptokinase (64.22%). In antimicrobial screening, the chloroform fraction displayed reasonable activity against most of the tested microorganisms. This study concludes that B. monnieri has potential antioxidant, anti-inflammatory and thrombolytic activities. Dhaka Univ. J. Pharm. Sci. 22(2): 155-161, 2023 (December)

Characterization of Nanoprecipitated PET Nanoplastics by 1H NMR and Impact of Residual Ionic Surfactant on Viability of Human Primary Mononuclear Cells and Hemolysis of Erythrocytes.

Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 μg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.

Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis

AbstractCell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Cryoprotectants for red blood cells: Evaluate safety and effectiveness by in vitro measures

AbstractThe transfusion of red blood cells (RBCs) is crucial in current medicinal research. The shelf‐life of donated RBCs preserved by the normal method is very short, limiting their clinical application. Cryopreservation is a reliable and effective technology for the long‐term storage of RBCs. During the process, ice formation will cause irreversible injuries to RBCs. Glycerol is used as a cryoprotectant (CPA) for RBCs to mitigate cryoinjuries. But it severely induces RBC hemolysis and deformation. This review introduces some biocompatible and effective CPAs used for RBC cryopreservation, outlining recent advances. Currently, there is no uniform standard to determine whether a CPA is suitable for RBCs. According to previous studies, we summarize for the first time comprehensive methods to evaluate the performance of CPAs by ensuring their safety and effectiveness. The safety of CPAs is defined as the degree of damage to RBCs, while the effectiveness is demonstrated by the properties of thawed RBCs, including membrane properties, protein activities, rheological properties, and metabolites levels. A novel CPA that is confirmed suitable for RBCs by these methods may be applied to other cells. We believe comprehensive methods can thoroughly evaluate the performance of CPAs and promote the development of transfusion therapy in clinic.

Modulation of Heme-Induced Inflammation Using MicroRNA-Loaded Liposomes: Implications for Hemolytic Disorders Such as Malaria and Sickle Cell Disease.

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.

Single Cell Analysis and Functional Features of Circulating CD34 + Cells from Patients with Sickle Cell Anaemia Unveil a Defect in the Myeloid Lineage

Polymorphonuclear neutrophils (PMN) have long been suspected to be involved in the pathophysiology of Sickle cell anemia (SCA) since early studies have reported a higher neutrophil count in patients at steady-state and its positive correlation with SCA severity. This high leukocyte count has long be considered as a peripheral phenomenon, driven by RBC hemolysis causing systemic inflammation and neutrophils recruitment. Nevertheless, several lines of evidence suggest that hematopoietic stem/progenitor cell (HSPC) function might be perturbed in SCA. Given that recent report showed an increased apoptosis during terminal erythroid maturation of SCA cells and that erythroid and myeloid differentiation derive from the same common myeloid progenitor (CMP), we investigated a possible enhanced myelopoiesis in SCA and its causes. First, to better understand when and how hyperleukocytosis and chronic inflammation begin in these patients, we analyzed longitudinal biological data from 1250 infants with SCA aged 1-24 months. We observed that the increase in neutrophils count began as early as the first months of life, and was statistically correlated with the drop in HbF rate and with the increase in reticulocytes count, much more than with the increase of the LDH level. These results seem to contradict the hypothesis of chronic inflammation driven by intravascular hemolysis. To assess whether the abnormal hematopoiesis is associated with a defect on lineage commitment of Hematopoietic Stem/Progenitor Cells (HSPCs), we evaluated the phenotype of myeloid and erythroid progenitors isolated from peripheral blood (PB) of untreated patients with SCA. While the portion of the common myeloid progenitor (CMP) was similar among both groups, we observed a significant increase of granulocyte-monocyte progenitors (GMP) in SCA patients (n=6) compared to health donors (HDs) (n=9), as well as a significant decrease of erythroid progenitors (MEP). Interestingly, HSPCs from SCA PB produced more myeloid colonies in vitro than those in HD HSPCs. In addition, colony-forming assay of sorted SCA CMP displayed an increase of granulo-monocytes colonies CFU-GM and decrease of erythroid colonies BFU-E compared to HDs, indicating an increased myeloid commitment of SCA HSPCs. To comprehensively characterize HSPC composition, we performed 10´ scRNA-seq on CD34+ HSPCs isolated from HDs (n=3) and patients with SCA (n=4). We confirmed that SCA HSPCs have a high proportion of neutrophil progenitors. More interestingly, MPP/HSC populations from SCA patients highly express several genes involved in myeloid differentiation like SELL (L-Selectin) and CSF3R (G-CSF receptor) genes, indicating an enhanced myeloid commitment of early progenitor in SCA. To better characterize the myeloid features of these progenitors, we investigated ex- vivo granulopoiesis of PB HSPCs from SCA patients (n=10) and HDs (n=9) in presence of G-CSF as CSF3R is highly expressed in early SCA progenitors. While cell proliferation was similar among both groups, unexpectedly SCA myeloid cells presented a significant delayed expression of CD11b (p<0.01). Interestingly, we also observed an overexpression of monocytic markers HLA-DR and CD36 in a subset of differentiated cells, suggesting a potential commitment of part of the myeloid progenitors to a monocyte lineage , confirmed by MGG staining. To further understand this defect, we performed RNAseq analysis of the GMP isolated from PB of SCA patients (n=4) and HD (n=4). We identified a total of 420 differentially expressed genes (DEGs). Among these DEGs, significantly downregulated genes in SCA included granulocyte differentiation program such as GFI1, C/EBPa, and JAK3. In return, significantly upregulated genes included KLF4, supporting the monocytic bias observed in the early myeloid progenitors. In conclusion, these data reveal the myeloid-primed signature in PB HSPCs of patients with SCA, likely responsible for the elevated PMN and monocyte count in these patients. This initial breakthrough in the understanding of the development of hyperleukocytosis and chronic inflammation in these patients will open up new early therapeutic options. Moreover, since CD34+ cells of the peripheral blood are the main source of HSPCs for the development of gene therapy in SCA, deciphering their functional feature and myelo-monocytic skewing could be of interest to guide the gene editing strategy.

Alternative Pathway Masp-3 Inhibitor OMS906 Effectively and Potently Inhibits Complement-Mediated Hemolysis in Preclinical Models Mechanistically Similar to Paroxysmal Nocturnal Hemoglobinuria

Introduction: Mannan-binding lectin-associated serine protease-3 (MASP-3) activates pro-complement Factor D (CFD) to form mature CFD, a rate-limiting enzyme in the alternative pathway (AP) of the complement system. CFD controls cleavage of complement Factor B (CFB) to form AP C3 convertase, which drives an amplification loop of protease complexes that activates both C3b-mediated opsonization and the terminal pathway. OMS906 is a humanized IgG4 monoclonal antibody (mAb) that binds to and inhibits MASP-3, thereby blocking maturation of CFD and downstream AP activity. Data from mice and monkeys demonstrated that OMS906 is highly selective and effectively inhibits AP activity. MASP-3 inhibition could provide therapeutic benefit in a variety of AP-mediated diseases, such as paroxysmal nocturnal hemoglobinuria (PNH). In PNH, a rare and life-threatening disorder, red blood cells (RBCs) lacking CD59 and CD55 regulatory surface proteins are targeted for rapid clearance via intravascular hemolysis and extravascular hemolysis, due to AP dysregulation. Clinical efficacy of OMS906 in a study of patients with PNH has been reported. To provide mechanistic support, we conducted in vitro and in vivo studies using preclinical models of complement-mediated hemolysis that have mechanistic overlap with PNH. Methods: In vitro potency of OMS906 was assessed using RBCs deficient in CD55 and CD59 to mimic the physiological effect of PNH. Healthy human donor RBCs were treated with inhibitory CD55 and CD59 antibodies, then incubated under AP assay conditions with human serum (diluted to 50%) containing OMS906, anti-C5 IgG4 mAb, or an isotype mAb control. The potency of OMS906, expressed as the half-maximal inhibitory concentration (IC 50), was assessed based on prevention of hemolysis of the PNH-like RBCs in vitro. Lysis was quantified by measuring hemoglobin released into sample supernatants using spectrophotometric absorbance. The in vivo effect of OMS906 on survival of murine RBCs prone to rapid clearance was assessed using RBCs from Crry -/- mice, which lack a rodent-specific complement regulatory protein that blocks AP activation. C57BL/6J male mice were intravenously injected with fluorescent labelled Crry -/- RBCs and received OMS906 or isotype mAb control via subcutaneous injection, or anti-CFB mAb via intraperitoneal injection. Blood samples were taken daily until Day 14 and the remaining number of Crry -/- RBCs measured by flow cytometry. Results: In vitro, OMS906 inhibited terminal cell lysis in human PNH-like RBCs more potently than a C5 inhibitor. Comparison of representative drug concentration-response showed that the average functional potency was 58-fold greater with OMS906 (IC 50: 0.8 μg/mL; 3 experiments; 12 total RBC donors) than with anti-C5 mAb (IC 50: 46.2 μg/mL; 2 experiments; 4 total RBC donors). In addition, the maximal extent to which lysis was inhibited was consistently greater with OMS906 than with anti-C5 mAb. In vivo, the rate of decline in number of Crry -/- RBCs was significantly slowed in mice treated with OMS906, in contrast to the rapid loss observed in isotype mAb control-treated mice ( Figure). OMS906 recipients showed a rate of Crry -/- RBC survival similar to the anti-CFB mAb group. Conclusion: OMS906 inhibited RBC lysis more potently than a C5 inhibitor, providing evidence that MASP-3 inhibition blocks downstream terminal activity and thus intravascular hemolysis. In addition, OMS906 improved survival of Crry -/- RBCs comparably to a CFB inhibitor, indicating that MASP-3 inhibition prevents the AP-mediated destruction of RBCs predictive of extravascular hemolysis. OMS906 is currently in clinical development for the treatment of PNH.

Deciphering Metabolomic Signatures Associated with Sickle Cell Disease Using Mouse Models

Introduction: Sickle cell disease (SCD) is debilitating and affects millions of people worldwide. Although SCD is caused by a single-point mutation of the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous, and there is a lack of well-characterized predictive biomarkers of disease progression. Metabolomics is a promising tool to interrogate disease pathophysiology and underlying molecular mechanisms. Although prior metabolomic studies have been performed on human and murine red blood cells (RBCs) in the context of SCD, no systematic investigation across commonly used mouse models of SCD has been published. In the present study, we examined the blood cell metabolome of 2 widely used SCD mouse models to study and compare the metabolomic changes in disease models. Methods: Blood samples were collected from 5 groups of mice (10-14 weeks old, n=6-7 per group): wild-type (WT) C57BL/6J mice; Townes AA, AS, and SS mice; and Berkeley SCD mice. Direct injection high-resolution mass spectrometry was used to generate untargeted metabolomics data. Three matrices (RBC, plasma, and whole blood) were isolated from each mouse group. The RBC fraction, containing peripheral blood mononuclear cells, was separated from plasma via centrifugation of whole blood. The current study focuses on the RBC dataset.Raw profile data were centroided, merged, and recalibrated using methods previously described (Fuhrer et al. Anal Chem 2011). Putative annotations were generated based on compounds contained in the Human Metabolome Database, KEGG, and ChEBI databases using accurate mass per charge (tolerance 0.001 m/z) and isotopic correlation patterns. Z-score normalization was applied across all samples for unsupervised clustering using principal component analysis (PCA) and to identify metabolites with significant changes between groups. Metabolites were mapped to central RBC metabolic pathways using MetaboAnalyst (Xia et al. Nat Protoc 2011) and manual curation. Results: Unsupervised PCA that used all detectable RBC metabolites resulted in clear separation of mouse groups by disease status (Figure 1), suggesting robust metabolomic changes in mouse models of SCD. Focusing on the top divergent metabolites across the mouse groups, several enriched pathways were identified, such as amino acid metabolism, pyrimidine and purine metabolism, and aminoacyl-tRNA biosynthesis, all potentially attributable to the buildup of RBC precursors. Metabolites characteristic of immature RBCs were further examined, with elevated hexokinase activity (hexose:hexose-6-phosphate ratio), ATP levels, and mitochondrial metabolites found in RBCs from SCD, in line with the significantly elevated reticulocyte content in SCD mice. Metabolites were curated from additional relevant RBC metabolic pathways; some showed significant differences among mouse groups, and others were significantly elevated in sickle mice (Townes SS and Berkeley SCD) compared with non-SCD controls (WT and Townes AA), including some glycolytic and pentose phosphate pathway (PPP) intermediates (Figure 2). The product-to-substrate ratios of metabolite levels from glycolytic and oxidative PPP (oxPPP) steps suggested glycolysis was favored and the later stage of the oxPPP was reduced in mouse models of SCD. There was also a higher metabolic turnover of glutathione, which is characteristic of SCD in humans, but we observed divergent metabolomic signatures between the disease models in our study (Figure 2). Conclusions: To our knowledge, this is the most comprehensive metabolomics study in mouse models of SCD. It provides a foundation to assess disease biology and response to treatment in future studies. Untargeted RBC metabolomics revealed metabolomic signatures that are consistent with human disease pathology in both murine SCD models, including reticulocytosis, hemolysis, and mitochondria retention in RBCs, and signatures that are unique to the murine models. Of the 2 main RBC metabolic pathways, glycolysis was favored over the oxPPP in SCD mice. This is the first step towards understanding the impact of SCD on RBC carbohydrate metabolism.

A Miniaturized, Rapid Technique for Measuring Red Blood Cell-Mediated Microcapillary Occlusion in HbAS, HbSC, and HbSS Sickle Cell Disease Genotypes

Introduction Sickle cell disease (SCD) is caused by a genetic mutation that produces sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, causing red blood cells (RBCs) to become sickle-shaped and poorly deformable. Stiff sickle RBCs contribute to microvascular occlusion and organ damage. Previous studies have developed microfluidic devices for assessing RBC-mediated occlusion under hypoxia, but the complexity of the setup and data acquisition limits their adoption. A rapid, easy-to-use, point-of-care assay for assessing RBC-mediated occlusion could increase throughput and aid in determining outcomes of novel therapies, including gene therapies, for SCD. Here, we induced RBC sickling using a chemical method in a simplified workflow to study hypoxic RBC-mediated microcapillary occlusion for healthy (HbAA), sickle cell trait (HbAS), hemoglobin SC disease (HbSC), and homozygous SCD (HbSS) RBCs. Additionally, a custom miniaturized impedance analyzer (MIA) was developed to provide a rapid and fully electronic measurement of occlusion within 10 minutes of sample perfusion. Methods Microfluidic devices were fabricated using standard photolithography and polydimethylsiloxane (PDMS) micro-molding protocols. Glass slides were coated with gold electrodes by sputter deposition and bound to the PDMS microfluidic devices using oxygen plasma. The microfluidic devices comprised six microcapillary arrays of widths 12, 10, 8, 6, 4, and 3 μm, with each array coupled with a pair of gold electrodes [3]. The MIA included an ADALM2000 software-defined instrument module, a custom-designed front-end interface board, and a Raspberry Pi single-board computing module. The ADALM2000 was used to generate and acquire a 10-kHz measurement signal, while the front-end interface board handled signal amplification and multiplexed signal routing through the microcapillary arrays. A custom graphical user interface on the Raspberry Pi allowed users to set experimental parameters and monitor data in real time. Venous blood samples were collected in EDTA tubes from participants with HbAS (n=9), HbSC (n=9), HbSS (n=13), and HbAA (n=6) under an IRB-approved protocol. To induce hypoxia, washed RBCs were suspended at 20% hematocrit in a 1.5% (w/v) sodium metabisulphite in 1× PBS buffer and incubated for 3 minutes at room temperature. For normoxia, RBCs from the same donors were suspended at 20% hematocrit in PBS and incubated with the same conditions. Samples were then perfused through the microfluidic device at a constant inlet pressure. After 10 minutes of perfusion, the change in electrical impedance across each array was used to calculate an occlusion index (OI), representing the percent occlusion of the capillary network. Results Normoxic OI for HbSS and HbSC was significantly higher than HbAA but there were no statistical differences in HbAA vs. HbAS and HbSC vs. HbSS (Figure 1A). HbAS, HbSC, and HbSS RBCs had significantly higher occlusion under hypoxia than in normoxia (Figure 1A, 1.46 ± 1.67% vs. 8.43 ± 2.78%, P = 0.004 for HbAS, 4.56 ± 2.77% vs. 27.1 ± 17.0%, P = 0.004 for HbSC, and 8.01 ± 6.26% vs. 58.3 ± 26.2%, P = 0.0002 for HbSS). Occlusion of HbAA RBCs remained low in both normoxia and hypoxia (2.70 ± 0.643% vs. 4.72 ± 0.953%, P = 0.0625). Hypoxic OI was significantly different between HbAA vs. HbAS (P = 0.017), HbAS vs. HbSC (P = 0.004), and HbSC vs. HbSS (P = 0.007). Hypoxic OI in HbSC and HbSS was associated with elevated markers of RBC hemolysis - absolute reticulocyte count (ARC, P = 0.0061) and lactate dehydrogenase (LDH, P < 0.0001, Figure 1B). Conclusion In normoxia, HbSC had comparable OI to HbSS likely due to HbC induced efflux of potassium ions and water resulting in cellular dehydration and poor RBC deformability. The hypoxic assay, however, unmasked the greater severity of HbSS. Further, elevated hypoxic OI in HbAS may be linked to higher risk of organ damage in this genotype. Chemically-induced hypoxia combined with electrical impedance-based measurement of RBC-mediated microcapillary occlusion offers a new technique for rapidly assessing RBC health and function in a low-oxygen environment. This approach eliminates the need for expensive, high-resolution microscopes and complex gas-exchange chambers. This may serve as a new standard for assessing the clinical efficacy of new treatments that improve RBC deformability and identifying at-risk patients in otherwise mild SCD genotypes.

Antioxidant, anti-inflammatory, antimicrobial, and anticancer properties of green broad bean pods (Vicia faba L.)

This study featured broad/fava bean pods as by-products of food production. It assessed the chemical composition of green bean pods (Vicia faba L.) and their methanolic extract.
 The extract was tested in vitro for antioxidant, anti-inflammatory, antimicrobial, and anticancer activities against prostate cancer (Pc3) and liver cancer (HepG2) cells. Broad bean pods proved to be rich in carbohydrates, fiber, protein, potassium, calcium, and magnesium. The extract contained 286 mg GAE/g total phenols and 105 mg QE/g total flavonoids. The antioxidant activity of the methanolic extract was measured by 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. The highest DPPH scavenging activity belonged to the extract concentrations of 1000 μg/mL (80.5%) and 500 μg/mL (73.7%), whereas the IC50 value was 87.35 μg/mL. The methanolic extract possessed the anti-inflammatory effect as it significantly reduced the hemolysis of red blood cells. The maximal inhibition percentage reached 66.7% at 1000 μg/mL. Regarding the antimicrobial activity, the broad bean pod methanolic extract inhibited Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, as well as Candida albicans. The extract reduced the cell viability of human hepatocarcinoma (HepG2) and prostate cancer (PC3) cells in a concentration-dependent manner. It also caused significant changes in cell shape, compared to the control.
 Therefore, broad beans can be recommended for human consumption together with pods, fresh or cooked, as a potential source of bioactive substances in functional food production.

Esterase-Responsive Polyglycerol-Based Nanogels for Intracellular Drug Delivery in Rare Gastrointestinal Stromal Tumors.

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels-Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC50 values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system.

Complement dysregulation associated with a genetic variant in factor H-related protein 5 in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) can be associated with mutations, deletions, or hybrid genes in factor H-related (FHR) proteins. A child with aHUS was investigated. Genetics was assessed by Sanger and next generation sequencing. Serum FHR5 was evaluated by immunoblotting, ELISA, and by induction of rabbit red blood cell hemolysis in the presence/absence of recombinant human rFHR5. Mutagenesis was performed in HEK cells. A heterozygous genetic variant in factor H-related protein 5 (CFHR5), M514R, was found in the child, who also had a homozygous deletion of CFHR3/CFHR1, and antibodies to factor H, as well as low levels of C3. Patient serum exhibited low levels of FHR5. In the presence of rabbit red blood cells, patient serum induced hemolysis which decreased when rFHR5 was added at physiological concentrations. Similar results were obtained using serum from the father, bearing the CFHR5 variant without factor H antibodies. Patient FHR5 formed normal dimers. The CFHR5 M514R variant was expressed in HEK cells and minimal secretion was detected whereas the protein level was elevated in cell lysates. Decreased secretion of the product of the mutant allele could explain the low FHR5 levels in patient serum. Reduced hemolysis when rFHR5 was added to serum suggests a regulatory role regarding complement activation on red blood cells. As such, low levels of FHR5, as demonstrated in the patient, may contribute to complement activation.

An operationally simple, one‐pot, convenient synthesis, and in vitro anti‐inflammatory activity of some new spirotriazolotriazine derivatives

AbstractThe wide biological actions of triazolotriazine hybrids, benzene‐sulfonamide derivatives, exhibit a good chance of displaying in vitro anti‐inflammatory effects. An operationally simple one‐pot, three‐component, and convenient synthesis method is used toward the synthesis of a series of diverse 2‐(2‐(4‐amino)‐1,3,5‐triazaspiro‐1,4‐dien‐2‐yl) hydrazinyl)‐N‐(4‐sulfamoylphenyl)‐2‐thioxoacetamides, which generated via reaction of 2‐hydrazinyl‐N‐(4‐sulfamoylphenyl)‐2‐thioxoacetamide, cyanoguanidine, and cyclic/acyclic ketones and were characterized by spectral analysis (IR, Nuclear Magnetic Resonance: 1H‐NMR, 13C‐NMR, and elemental analysis). The spirotriazolotriazine derivatives are obtained with up to 95% yield, and their structure–activity relationship was discussed. All compounds were tested for in vitro anti‐inflammatory potential using the inhibition of RBC hemolysis technique and COX inhibition assay. The results from RBC hemolysis technique demonstrated that almost all evaluated compounds exhibited significant in vitro anti‐inflammatory efficacy. More specifically, compounds 7, 8, and 12 have superior membrane stability over indomethacin as a standard drug. At concentrations of 400, 600, 800, and 1000 μg/mL, compound 12 demonstrated highly significant inhibition of RBC hemolysis with 95.2%, 97.3%, 98.9%, and 99.8%, respectively, compared with indomethacin at the same concentrations (94.5%, 97.3%, 98.5%, and 99.3%, respectively). COX assay indicated that compounds 7, 8, and 12 exhibited excellent COX‐2 inhibition percentage (98.51 ± 0.01, 98 ± 0.01, and 98.97 ± 0.06, respectively) at 150 μg/mL using indomethacin (97.3 ± 0.005). Furthermore, an investigation using molecular docking against COX‐2 (pdb ID: 5IKT) was used to analyze the anti‐inflammatory properties of all studied substances. When compared to indomethacin, molecular docking research showed a strong connection with the intended protein and an elevated docking score. According to in vitro anti‐inflammatory action and computational approach, they show promise as a therapeutic candidate for the treatment of inflammatory diseases.

Genetic Polymorphisms in the Ferrireductase STEAP3 Regulate a Ferroptosis-like Process of Lipid Peroxidation-Induced Hemolysis in Murine and Human Red Blood Cells

Red blood cell (RBC) storage in the blood bank induces a series of biochemical and morphological alterations, collectively denoted as the storage lesion(s). Membrane damage by lipid peroxidation is a hallmark of the storage lesion, a process thought to be triggered by oxidant stress. To identify the genetics underlying this phenomenon, we leveraged a novel murine system, the Jackson lab Diversity Outbred (JDO) mouse colony, which was obtained by extensively cross-breeding 8 founder mouse strains with extreme genetically heterogeneity ( Figure 1.A). A total of 350 fresh and stored (up to 7 days) murine RBC samples were tested by mass spectrometry-based metabolomics and lipidomics. At the end of storage, post-transfusion recovery (PTR), which measures intra- and extravascular hemolysis and is a gold standard for determining RBC storage quality, was determined by quantifying the percentage of stored RBCs that were still circulating 24h after their transfusion into Ubi-GFP+ recipient mice ( Figure 1.A). Genome wide-association studies identified a strong association between PTR and a polymorphic region on chromosome 1 ( Figure 1.A), which encodes STEAP3, a ferrireductase. This region was associated with altered metabolism of stored RBCs ( Figure 1.A), especially an elevation of lipid peroxidation products (hive plot in Figure 1.A). Mechanistically, these data support a role for STEAP3 in promoting Fenton and Haber-Weiss chemistry in iron-loaded RBCs. Specifically, ferrous iron participates in this chemistry, becoming oxidized to its ferric state with concomitant generation of hydroxyl or hydroperoxyl radicals; these in turn attack fatty acids (preferentially, poly- and highly-unsaturated fatty acids, such as octadecadienoic and eicosatetraenoic acid), thereby promoting lipid peroxidation (to hydroxyoctadecadienoic (HODE) and hydroxyeicosatetraenoic (HETE) acids, respectively). By reducing Fe 3+ to Fe 2+, STEAP3 can shift the balance of this reaction by increasing the availability of the reactant, thereby favoring the generation of product radicals; this process is analogous to ferroptosis, as described in other biological systems. To extend the relevance of these findings to humans, we performed metabolomics on end-of-storage (i.e., Day 42) packed RBCs from 13,091 human donors in the Recipient Epidemiology and Donor Evaluation Study (REDS RBC Omics; Figure 1.B), enrolled in four blood centers across the United States. We stratified donors based on oxylipin levels (e.g., acids - HETEs - Figure 1.B) and common STEAP3 polymorphisms (e.g., a representative non-synonymous coding single nucleotide polymorphism (SNP), rs17013371, was observed with ~10% prevalence in these donors), along with 37 other common SNPs observed in this study ( Figure 1.B). When correlating metabolic and hematological parameters to common STEAP3 non-synonymous coding SNPs, we found strong associations of such polymorphisms with RBC susceptibility to hemolysis (either osmotic or oxidative), lipid peroxidation, RBC numbers, and hemoglobin levels ( Figure 1.B). These associations held true for a subset of the original donors with extreme hemolytic propensity (n=643) who donated a second RBC unit that was again stored for 42 (i.e., the “recalled donor population”). In the initial (n=13,091) and recalled donor (n=643) populations, lipid peroxidation products, HETEs, and HODEs ranked as the top predictors of hemolysis and vesiculation. In summary, we identified a role for the STEAP3 ferrireductase in contributing to a ferroptosis-like phenotype of lipid peroxidation that is associated with increased susceptibility to hemolysis in vitro and in vivo in murine and human RBCs following storage under blood bank conditions. Figure 1 - A. Jackson Lab diversity outbred (JDO) mice were bred from 8 founder mouse strains with extreme genetic heterogeneity. A total of 350 fresh and stored murine red blood cells (RBCs) were tested for metabolomics, lipidomics, and post-transfusion recovery (PTR). A polymorphic region on chromosome 1, coding for the ferrireductase STEAP3 was associated with heterogeneous PTR and elevation in lipid peroxidation products. B. Metabolomics analyses of 13,091 donors from the REDS RBC Omics identified an association between the end of storage levels of oxylipins and common polymorphisms for STEAP3, both linked to an increased RBC susceptibility to hemolysis (either osmotic or oxidative.

HbF Inducing Eed Inhibitor Ftx-6058 Selectively and Reversibly Impacts Bone Marrow in Wild-Type Mice

Sickle cell disease (SCD) is a genetic disorder characterized by red blood cell sickling, hemolysis, vaso-occlusive crises (VOCs), and other complications resulting from a mutation in the HBB gene. Increasing fetal hemoglobin (HbF) levels has shown promise in mitigating disease-related pathophysiology. In individuals with SCD who exhibit hereditary persistence of fetal hemoglobin (HPFH) and have HbF levels ranging from 20% to 30%, a remarkable reduction in symptoms is observed, highlighting the protective effect of increased HbF in SCD. Preclinical and clinical findings indicate that compounds inhibiting the embryonic ectoderm development (EED) component of polycomb repressive complex 2 (PRC2) leads to elevations in HbF to therapeutic levels. These results suggest the potential for a substantial benefit for individuals living with SCD. To further understand the effects of PRC2 inhibition, this study aims to characterize the onset and reversibility of EED inhibition by FTX-6058. Results from in vivo studies in wild-type mice demonstrated upregulation of the mouse fetal hemoglobin ortholog mRNA (Hbb-bh1) as well as target engagement in bone marrow though quantification of H3K27 trimethylation during seven days of active dosing of FTX-6058. Target engagement and differential gene expression profiles in the bone marrow subsequently returned to baseline shortly after the dosing period. These findings demonstrate that inhibition of EED through FTX-6058 leads to a minimal, transient impact on the bone marrow in mice, and any transcriptional changes are rapidly reversible following cessation of dosing. A small molecule EED inhibitor has the potential to provide a new treatment for sickle cell disease without irreversibly altering the bone marrow.

Simeprevir restores the anti-Staphylococcus activity of polymyxins

Methicillin-resistant Staphylococcus aureus (MRSA) infection poses a severe threat to global public health due to its high mortality. Currently, polymyxins are mainly used for the treatment of Gram-negative bacterial-related infection, while exhibiting limited antibacterial activities against Staphylococcus aureus (S. aureus). However, the combination of antibiotics with antibiotic adjuvants is a feasible strategy for the hard-treated infection and toxicity reducing. We will investigate the antibacterial activity of simeprevir (SIM), which treated for genotype 1 and 4 chronic hepatitis C, combined with polymyxins against MRSA through high-throughput screening technology. In our study, the synergistic antibacterial effect of SIM and polymyxins against S. aureus in vitro was found by checkerboard assay and time-growth curve. The cytotoxicity of SIM combined with polymyxin B sulfate [PB(S)] or polymyxin E (PE) in vitro was evaluated using CCK-8, human RBC hemolysis and scratch assays. In addition, we investigated the eradication of biofilm formation of S. aureus by biofilm inhibition assay and the killing of persister cells. Moreover, we evaluated the therapeutic effect and in vivo toxicity of the combination against MRSA in murine subcutaneous abscess model. Furthermore, it was preliminarily found that SIM significantly enhanced the destruction of MRSA membrane by SYTOX Green and DISC3(5) probes. In summary, these results reveal that the therapy of SIM combined with polymyxins (especially PE) is promising for the treatment of MRSA infection.

Transcriptome analysis of Corvus splendens reveals a repertoire of antimicrobial peptides

Multidrug resistance has become a global health problem associated with high morbidity and mortality. Antimicrobial peptides have been acknowledged as potential leads for prospective anti-infectives. Owing to their scavenging lifestyle, Corvus splendens is thought to have developed robust immunity to pathogens found in their diet, implying that they have evolved mechanisms to resist infection. In the current study, the transcriptome of C. splendens was sequenced, and de novo assembled to identify the presence of antimicrobial peptide genes. 72.09 million high-quality clean reads were obtained which were then de novo assembled into 3,43,503 transcripts and 74,958 unigenes. About 37,559 unigenes were successfully annotated using SwissProt, Pfam, GO, and KEGG databases. A search against APD3, CAMPR3 and LAMP databases identified 63 AMP candidates belonging to more than 20 diverse families and functional classes. mRNA of AvBD-2, AvBD-13 and CATH-2 were found to be differentially expressed between the three tested crows as well as among the tissues. We also characterized Corvus Cathelicidin 2 (CATH-2) to gain knowledge of its antimicrobial mechanisms. The CD spectroscopy of synthesized mature Corvus CATH-2 peptide displayed an amphipathic α-helical structure. Though the synthetic CATH-2 caused hemolysis of human RBC, it also exhibited antimicrobial activity against E. coli, S. aureus, and B. cereus. Docking simulation results revealed that this peptide could bind to the LPS binding site of MD-2, which may prevent LPS from entering the MD-2 binding pocket, and trigger TLR4 signaling pathway. The Corvus CATH-2 characterized in this study could aid in the development of novel therapeutics.

Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

Aim. In this study, blood compatibility of ZnO nanoparticles-polymer nanocomplex (D-PAA/ZnONPs()) synthesized in situ into dextran-graft-polyacrylamide (D-PAA) using zinc sulphate as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays. Materials and methods. Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0–800 mg l−1 analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs() nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs() nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca2+-mediated mechanisms in D-PAA/ZnONPs() nanocomplex-induced erythrotoxicity. Results. Hemocompatibility assessment of the D-PAA/ZnONPs() nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg l−1, respectively. Oxidative stress- or Ca2+-mediated mechanisms were not involved in D-PAA/ZnONPs() nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs() nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs() nanocomplex did not induce eryptosis in vitro. Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials. Conclusions. The D-PAA/ZnONPs() nanocomplex has a good hemocompatibility profile at low concentrations. Hemocompatibility testing in nanotoxicology should include both eryptosis and hemolysis assays.