Ginsenoside Rh2 (GRh2) exhibits significant potential as an anticancer agent; however, progress in developing chemotherapeutic drugs is impeded by their toxicity toward off-target tissues. Specifically, anemia caused by chemotherapy is a debilitating side effect and can be caused by red blood cell (RBC) hemolysis and eryptosis. Cells were exposed to GRh2 in the antitumor range and hemolytic and eryptotic markers were examined under different experimental conditions using photometric and cytofluorimetric methods. GRh2 caused Ca2+-independent, concentration-responsive hemolysis in addition to disrupted ion trafficking with K+ and Cl- leakage. Significant increases in cells positive for annexin-V-fluorescein isothiocyanate, Fluo4, and 2,7-dichlorofluorescein were noted upon GRh2 treatment coupled with a decrease in forward scatter and acetylcholinesterase activity. Importantly, the cytotoxic effects of GRh2 were mitigated by ascorbic acid and by blocking casein kinase 1α (CK1α) and mixed lineage kinase domain-like (MLKL) signaling. In contrast, Ca2+ omission, inhibition of KCl efflux, and isosmotic sucrose aggravated GRh2-induced RBC death. In whole blood, GRh2 selectively targeted reticulocytes and lymphocytes. Altogether, this study identified novel mechanisms underlying GRh2-induced RBC death involving Ca2+ buildup, loss of membrane phospholipid asymmetry and cellular volume, anticholinesterase activity, and oxidative stress. These findings shed light on the hematologic toxicity of GRh2 which is crucial for optimizing its utilization in cancer treatment.

The SP/KLF family of transcription factors harbour three C-terminal C2H2 zinc fingers interspersed by two linkers which confers DNA-binding to a 9-10 bp motif. Mutations in KLF1, the founding member of the family, are common. Missense mutations in linker two result in a mild phenotype. However, when co-inherited with loss-of-function mutations, they result in severe non-spherocytic hemolytic anemia. We generate a mouse model of this disease by crossing Klf1+/- mice with Klf1H350R/+ mice that harbour a missense mutation in linker-2. Klf1H350R/- mice exhibit severe hemolysis without thalassemia. RNA-seq demonstrate loss of expression of genes encoding transmembrane and cytoskeletal proteins, but not globins. ChIP-seq show no change in DNA-binding specificity, but a global reduction in affinity, which is confirmed using recombinant proteins and in vitro binding assays. This study provides new insights into how linker mutations in zinc finger transcription factors result in different phenotypes to those caused by loss-of-function mutations.

BACKGROUND: Pulsed-field ablation (PFA) of atrial fibrillation (AF) is a new method in clinical practice. Despite a favorable safety profile of PFA in AF ablation, rare cases of renal failure, probably due to hemolysis, have been recently reported.
OBJECTIVE: The aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities.
METHODS: Blood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation (IRE) using 216 bipolar pulses, each lasting 2 μs with 5 μs intervals, repeated 20 times at a frequency of 1 Hz. These pulses varied in from 500 to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles (RBCμ) were evaluated using flow cytometry. Cardiomyocyte death was quantified using propidium iodide.
RESULTS: PF energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.31 ± 0.16 g/l, 2.33 ± 0.90 g/l, and 5.7 ± 0.20 g/l, p< 0.05), and similar increase in the concentration of RBCμ. Significant rates of cardiomyocyte death were observed at electric field strengths of 750 V/cm, 1000 V/cm, 1250 V/cm and 1500 V/cm (26.5 ± 5.9%, 44.3 ± 6.2%, 55.5 ± 6.9% and 74.5 ± 17.8% of cardiomyocytes, p < 0.05).
CONCLUSIONS: The most effective induction of cell death in vitro was observed at 1500 V/cm. This intensity was also associated with a significant degree of hemolysis.

Unfortunately, during pathological conditions resulting in chronic hemolysis cell-free hemoglobin (Hb) is released into the circulation which releases free heme, resulting in several complications. One approach to prevent these toxicities is administration of supplemental scavenger proteins, haptoglobin (Hp) and hemopexin (Hpx). The goal of this body of work is to objectively measure levels of vascular reactivity and inflammatory profiles after an infusion of acellular hemoglobin in animals that were given a co-administration of PEGylated human apohemoglobin (PEG-apoHb), a hemopexin (Hpx)-mimetic that can scavenge free heme from hemoglobin, together with human plasma-derived Hp that can scavenge dimerized Hb. Utilizing intravital microscopy, Golden Syrian hamsters instrumented with a dorsal window chamber were used to evaluate the in vivo effects of 4 experimental groups that were then challenged with a hypovolemic injection (10% of the animal\'s blood volume) of human Hb (hHb, 5 g/dL). The four experimental groups consisted of: 1) lactated Ringer\'s (control), 2) PEG-apoHb only, 3) Hp only, and 4) PEG-apoHb + Hp. The microvascular hemodynamics (diameter and flow) in arterioles and venules were recorded at baseline, 20 minutes after treatment, and 20 minutes after hHb challenge. Systemic parameters (blood pressure and heart rate), blood gases (pH, pCO2, and pO2), blood parameters (Hb concentration and hematocrit), and multiorgan functionality/ inflammation were also measured. Our results suggest that co-administration of PEG-apoHb + Hp as a booster prior to the infusion of acellular hemoglobin significantly prevented vasoconstriction in the microcirculation, significantly increased the number of functional capillaries, and significantly reduced inflammation.

Infections by drug-resistant microorganisms are a threat to global health and antimicrobial peptides are considered to be a new hope for their treatment. Temporin-WY2 was identified from the cutaneous secretion of the Ranidae frog, Amolops wuyiensis. It presented with a potent anti-Gram-positive bacterial efficacy, but its activity against Gram-negative bacteria and cancer cell lines was unremarkable. Also, it produced a relatively high lytic effect on horse erythrocytes. For further improvement of its functions, a perfect amphipathic analogue, QUB-1426, and two lysine-clustered analogues, 6K-WY2 and 6K-1426, were synthesised and investigated. The modified peptides were found to be between 8- and 64-fold more potent against Gram-negative bacteria than the original peptide. Additionally, the 6K analogues showed a rapid killing rate. Also, their antiproliferation activities were more than 100-fold more potent than the parent peptide. All of the peptides that were examined demonstrated considerable biofilm inhibition activity. Moreover, QUB-1426, 6K-WY2 and 6K-1426, demonstrated in vivo antimicrobial activity against MRSA and E. coli in an insect larvae model. Despite observing a slight increase in the hemolytic activity and cytotoxicity of the modified peptides, they still demonstrated a improved therapeutic index. Overall, QUB-1426, 6K-WY2 and 6K-1426, with dual antimicrobial and anticancer functions, are proposed as putative drug candidates for the future.

Heme is essential for a variety of proteins involved in vital physiological functions in the body, such as oxygen transport, drug metabolism, biosynthesis of steroids, signal transduction, antioxidant defense and mitochondrial respiration. However, free heme is potentially cytotoxic due to the capacity of heme iron to promote the oxidation of cellular molecules. The liver plays a central role in heme metabolism by significantly contributing to heme synthesis, heme detoxification, and recycling of heme iron. Conversely, enzymatic defects in the heme biosynthetic pathway originate multisystemic diseases (porphyrias) that are highly associated with liver damage. In addition, there is growing evidence that heme contributes to the outcomes of inflammatory, metabolic and malignant liver diseases. In this review, we summarize the contribution of the liver to heme metabolism and the association of heme dyshomeostasis with liver disease.

Pigment-induced acute kidney injury (AKI) is an important and preventable complication of rhabdomyolysis or hemolysis. It is characterized by the release of free heme pigment (myoglobin or hemoglobin) in the circulation, leading to direct injury of the proximal tubule and distal tubule obstruction by pigment cast. We are reporting eight cases of pigment-induced AKI, including six cases of myoglobin cast nephropathy and two cases of hemoglobin cast nephropathy. The causes of rhabdomyolysis were strenuous exercise, infection/febrile illness, and drug-induced neuroleptic malignant syndrome. Paroxysmal nocturnal hemoglobinuria and anti-tuberculosis treatment (rifampicin and isoniazid) had led to hemoglobin cast nephropathy each in one case. Seven cases had severe renal failure requiring dialysis. Short-term renal outcome was favorable. However, long-term follow-up is necessary to determine whether pigment-induced AKI has delayed sequelae. Therefore, clinicians should consider rhabdomyolysis or hemolysis as potential hidden causes of AKI in diverse clinical conditions, especially those of non-traumatic origin, to achieve an accurate diagnosis.

This study evaluates the antiproliferative potential of flavanones, chromanones and their spiro-1-pyrazoline derivatives as well as their inclusion complexes. The main goal was to determine the biological basis of molecular pro-apoptotic activities and the participation of reactive oxygen species (ROS) in shaping the cytotoxic properties of the tested conjugates. For this purpose, changes in mitochondrial potential and the necrotic/apoptotic cell fraction were analyzed. Testing with specific fluorescent probes found that ROS generation had a significant contribution to the biological anticancer activity of complexes of flavanone analogues. TT (thrombin time), PT (prothrombin time) and APTT (activated partial tromboplastin time) were used to evaluate the influence of the compounds on the extrinsic and intrinsic coagulation pathway. Hemolysis assays and microscopy studies were conducted to determine the effect of the compounds on RBCs.

Intravascular hemolysis is a central feature of congenital and acquired hemolytic anemias, complement disorders, infectious diseases, and toxemias. Massive and/or chronic hemolysis is followed by the induction of inflammation, very often with severe damage of organs, which enhances the morbidity and mortality of hemolytic diseases. Galectin-3 (Gal-3) is a β-galactoside-binding lectin that modulates the functions of many immune cells, thus affecting inflammatory processes. Gal-3 is also one of the main regulators of fibrosis. The role of Gal-3 in the development of different kidney and liver diseases and the potential of therapeutic Gal-3 inhibition have been demonstrated. Therefore, the objective of this review is to discuss the possible effects of Gal-3 on the process of kidney and liver damage induced by intravascular hemolysis, as well as to shed light on the potential therapeutic targeting of Gal-3 in intravascular hemolysis.

OBJECTIVE : The treatment of mitral valve prolapse involves two distinct repair techniques: chordal replacement (Neochordae technique) and leaflet resection (Resection technique). However, there is still a debate in the literature about which is the optimal one. In this context, we performed an image-based computational fluid dynamic study to evaluate blood dynamics in the two surgical techniques. METHODS : We considered a healthy subject (H) and two patients (N and R) who underwent surgery for prolapse of the posterior leaflet and were operated with the Neochordae and Resection technique, respectively. Computational Fluid Dynamics (CFD) was employed with prescribed motion of the entire left heart coming from cine-MRI images, with a Large Eddy Simulation model to describe the transition to turbulence and a resistive method for managing valve dynamics. We created three different virtual scenarios where the operated mitral valves were inserted in the same left heart geometry of the healthy subject to study the differences attributed only to the two techniques. RESULTS : We compared the three scenarios by quantitatively analyzing ventricular velocity patterns and pressures, transition to turbulence, and the ventricle ability to prevent thrombi formation. From these results, we found that the operative techniques affected the ventricular blood dynamics in different ways, with variations attributed to the reduced mobility of the Resection posterior leaflet. Specifically, the Resection technique resulted in turbulent forces, related with the risk of hemolysis formation, up to 640 Pa, while the other two scenarios exhibited a maximum of 240 Pa. Moreover, in correspondence of the ventricular apex, the Resection technique reduced the areas with low velocity to 15%, whereas the healthy case and the Neochordae case maintained these areas at 30 and 48%, respectively. Our findings suggest that the Neochordae technique developed a more physiological flow with respect to the Resection technique. CONCLUSION: Resection technique gives rise to a different direction of the mitral jet during diastole increasing the ability to washout the ventricular apex preventing from thrombi formation, but at the same time it promotes turbulence formation that is associated with ventricular effort and risk of hemolysis.