Significant Hemolysis Research Articles

Enhancing the antimycobacterial efficacy of pyridine-4-carbohydrazide: linkage to additional antimicrobial agents via oxocarboxylic acids.

This study evaluates the antimycobacterial potential of novel "mutual" bioactive amides, combining pyridine-4-carbohydrazide (isoniazid, INH) with various antimicrobial agents (sulphonamides, 4-aminosalicylic acid, thiosemicarbazide, diphenyl (thio)ethers) via oxocarboxylic acids. The aim was to enhance activity against both drug-susceptible and multidrug-resistant (MDR) Mycobacterium tuberculosis and non-tuberculous strains, while overcoming drug resistance through dual-action mechanisms. Many derivatives exhibited potent antimycobacterial activity, with minimum inhibitory concentrations (MICs) as low as ≤0.25 μM, outperforming INH, especially diphenyl (thio)ethers and biphenyl analogues. Additionally, the compounds were effective against M. kansasii (MICs ≤1 μM) and inhibited MDR strains at higher concentrations (≥8 μM). The cytotoxicity assay indicated a favourable safety profile, with no significant haemolysis at 125 μM, and some compounds were even protective. Selectivity for mycobacteria was confirmed by low inhibition of Gram-positive bacteria and inactivity against Gram-negative bacteria or fungi, highlighting the potential for further development as antimycobacterial agents.

Hemocompatible nucleosome-inspired heparin-mimicking hydrogel microspheres for safe and efficient extracorporeal removal of circulating histones in critically ill patients.

Circulating histones have been identified as essential mediators that lead to hyperinflammation, platelet aggregation, coagulation cascade activation, endothelial cell injury, multiple organ dysfunction, and death in severe patients with sepsis, multiple trauma, COVID-19, acute liver failure, and pancreatitis. Clinical evidence suggests that plasma levels of circulating histones are positively associated with disease severity and survival in patients with such critical diseases. However, safe and efficient therapeutic strategies targeting circulating histones are lacking in current clinical practice. Extracorporeal blood purification, a widely used life support technique in intensive care units, is a promising therapeutic option for eliminating circulating histones. Inspired by electrostatic interactions between DNA chains and histones in natural nucleosomes, we propose a "one stone kills two birds" strategy to combat histone-related critical diseases by developing heparin-mimicking hydrogel microspheres (RCHMs). On one hand, the heparin-mimicking hydrogel structure inside RCHMs contains a large number of carboxyl and sulphonic acid groups by in situ cross-linking polymerization, which endows the RCHMs with excellent hemocompatibility. On the other hand, the RCHMs can adsorb circulating histones through electrostatic interactions. Our results demonstrate that the RCHMs do not cause significant hemolysis, blood cell activation and complement activation, with improved anti-protein contamination properties. The tailored RCHM microspheres (A3M1) can efficiently and selectively adsorb 91.16% of calf thymus histones with an adsorption capacity of 20.47 μg mg-1 within 4 h. Moreover, the RCHMs significantly attenuate histone-mediated thrombocytopenia, platelet aggregation, and endothelial cell death. Therefore, the RCHMs are promising hemoperfusion adsorbents for extracorporeal removal of circulating histones from the blood of critically ill patients, providing a new insight into the management of multiple histone-related disorders.

Zeatin Elicits Premature Erythrocyte Senescence Through Calcium and Oxidative Stress Mediated by the NOS/PKC/CK1α Signaling Axis.

Objectives: Cytokinins are plant hormones that regulate cell growth and differentiation. In particular, zeatin (ZTN) delays cellular senescence of human fibroblasts and keratinocytes and exhibits anticancer activity. Chemotherapy-induced anemia is a major side effect of anticancer therapy secondary to premature senescence of red blood cells (RBCs). Herein, we investigated the biochemical and molecular mechanisms underlying ZTN action in human RBCs. Methods: Colorimetric assays were used to quantify hemolysis and related markers and flow cytometric analysis was applied to examine eryptosis through phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, reactive oxygen species (ROS) by H2DCFDA, and cell size from forward scatter (FSC). Results: ZTN at 200μM induced significant hemolysis and K+, Na+, AST, and LDH leakage. ZTN also caused a significant increase in annexin-V-positive cells along with increased Fluo4 and DCF fluorescence and reduced FSC. Importantly, L-NAME, staurosporin, D4476, urea, sucrose, and polyethylene glycol 8000 (PEG) significantly ameliorated ZTN cytotoxicity. Conclusion: ZTN stimulates PS exposure, intracellular Ca2+ elevation, oxidative stress, and cell shrinkage. The hemolytic potential of ZTN, mediated through nitric oxide synthase/protein kinase C/casein kinase 1α signaling axis, is sensitive to isosmotic urea, sucrose, and PEG availability. Altogether, the anticancer potential of ZTN must be reconsidered with prudence.

Long-Term Efficacy and Safety of Danicopan as Add-On Therapy to Ravulizumab or Eculizumab in PNH With Significant EVH.

Complement C5 inhibitor treatment with ravulizumab or eculizumab for paroxysmal nocturnal hemoglobinuria (PNH) improves outcomes and survival. Some patients remain anemic due to clinically significant extravascular hemolysis (cs-EVH: hemoglobin [Hgb] ≤9.5 g/dL and absolute reticulocyte count [ARC] ≥120×109/L). In the phase 3 ALPHA trial, participants received oral factor D inhibitor danicopan (150 mg 3 times daily) or placebo plus ravulizumab or eculizumab during the 12-week, double-blind treatment period (TP) 1; those receiving placebo switched to danicopan during the subsequent 12-week, open-label TP2 and continued during the 2-year long-term extension (LTE). There were 86 participants randomized in the study, of whom 82 entered TP2 and 80 entered LTE. The primary endpoint was met, with Hgb improvements from baseline at week 12 (Wk12) (LS mean change: 2.8 g/dL) with danicopan. For participants switching from placebo to danicopan at Wk12, improvements in mean Hgb were observed at Wk24. Similar trends were observed for the proportion of participants with ≥2 g/dL Hgb increase, ARC, proportion of participants achieving transfusion avoidance, and Functional Assessment of Chronic Illness Therapy-Fatigue scale scores. Improvements were maintained up to week 72. No new safety signals were observed. The breakthrough hemolysis rate was 6 events per 100 patient-years. These long-term data demonstrate sustained efficacy and safety of danicopan plus ravulizumab/eculizumab for continued control of terminal complement activity, intravascular hemolysis, and cs-EVH in PNH. Registration: Clinicaltrials.gov, NCT04469465.

Quantification of hemolysis rates from pulsed field ablation

Abstract Introduction Pulsed electric fields induce hemolysis of red blood cells as a dose-dependent effect of the electric field. Voltage pulsations induce a transmembrane potential across the cell membrane and either open up or create pores in the red cells. In isotonic conditions, the pores allow passage of potassium and sodium ions but not sucrose or hemoglobin, and leakage of ions leads to an osmotic imbalance which in turn causes a colloidal hemolysis of the red cells. Objective To quantify hemolysis rates during pulsed field ablation (PFA). Methods We created a computational model of PFA to determine the total volume of blood hemolyzed from each pulse during catheter ablation using pulsed field energy. The percentage hemolysis was calculated as a function of electric field intensity utilizing existing published experimental data. A single electrode delivering a single pulse of 100 microseconds duration was modeled at voltages of 1 kV, 1.5 kV, and 2 kV. Results The total volume of hemolyzed blood per pulse increased with increasing applied voltage (Figure). For 2 electrodes in direct tissue contact, the electrode surface exposed to the blood pool generated 19 microliters of hemolyzed red blood cells with a single pulse of a 2 kV field strength. In the case of a single pulse train consisting of 50 pulses, using a catheter configuration with 20 electrodes, an estimate of total volume per pulse train is approximately 9.5 mL of hemolyzed blood. In the case of electrodes not in direct contact with endocardial tissue, and exposed to the blood pool, up to double this volume could be anticipated per pulse train. Hemolyzed volume can be reduced with better tissue contact (reducing exposure to blood), or potentially with the use of irrigation to dilute the local red blood cell concentration. Increased tonicity of the irrigant fluid may also reduce the osmotic pressure increases that result in hemolysis. Conclusions Typical voltages utilized in pulsed field ablation can cause significant hemolysis, which is exacerbated by inadequate tissue contact. A greater number of electrodes and number of pulses delivered increases the risk, whereas better tissue contact, and the use of irrigation, may reduce it.

Haemolysis and acute tubular injury after pulsed field ablation compared to radiofrequency: An observational study

Abstract Background Pulsed field ablation (PFA) represents a novel promising non-thermal alternative in non-pharmacological treatment of atrial fibrillation (AF). However, infrequent cases of acute renal failure secondary to intravascular haemolysis after a PFA procedure have been described. Objective To investigate the impact of ablation technology (PFA vs. radiofrequency ablation (RFA)) on the level of plasma cell-free haemoglobin (CFH) and the concentration of serum neutrophil gelatinase-associated lipocalin (NGAL) as a marker of tubular injury. Methods This was a prospective observational trial. In a consecutive cohort of patients who underwent AF ablation (PFA of RFA), blood samples were drawn just before the procedure (Sample 1: CFH and NGAL), immediately after the procedure (Sample 2: CFH) and one day after the procedure (Sample 3: CFH and NGAL). Plasma CHF level and serum NGAL concentration were analysed using the ELISA technique. Results Among 70 patients enrolled (mean age 64.3 ± 10.3 years, 61 % male), 23 underwent RFA and 47 PFA (22 pulmonary vein isolation (PVI) only and 25 PVI with other additive lesions). Baseline serum creatinine levels of both groups were comparable (91.7 ± 22.1 µmol/L vs. 88.8 ± 22.1 µmol/L P = 0.44). In the PFA cohort, a significant peak of CFH concentration was observed just after the procedure with a subsequent decrease within 12 hours (Sample 1 vs. Sample 2: 93.4 ± 65.1 µg/mL vs. 2394.9 ± 1966.1 µg/mL P < 0.001, Sample 1 vs. Sample 3: 93.4 ± 65.1 µg/mL vs. 97.2 ± 68.5 µg/mL P = 0.75). No significant differences were observed in the RFA cohort. Compared to baseline, neither the PFA nor the RFA group showed a significant difference in NGAL levels (PFA 98.6 ± 31.7 ng/mL vs. 98.5 ± 38.1 ng/mL P = 0.78, RFA 108.3 ± 33.8 ng/mL vs. 116.3 ± 32.2 ng/mL P = 0.49). Conclusions Compared to RFA, PFA leads to significant peri-procedural haemolysis. However, no differences in the concentration of the tubular injury marker NGAL were observed on the first day after the procedure.

Accuracy of the HemoCue® to measure cell-free plasma hemoglobin and detect clinically significant hemolysis.

Monitoring cell-free plasma hemoglobin (PHb) during extracorporeal therapies allows early intervention of significant hemolysis, but timely measurements are often challenging. We thus present an analysis of a rapid benchtop device's ability to detect clinically significant hemolysis (PHb ≥50mg/dL). PHb was measured in 419 plasma samples from 88 pediatric patients undergoing cardiopulmonary bypass via both the benchtop device (HemoCue® Plasma/Low Hb system) and the clinical laboratory at the Children's Hospital of Pittsburgh (reference standards). Values of PHb ≥50mg/dL as measured by the reference standard was defined as the binary outcome of clinically significant hemolysis. Analyses included Pearson correlations, logistic regression, receiver operating characteristic curves, and Bland-Altman. Because the manufacturer specifications identify the measurement range of the HemoCue® system as 30-3000mg/dL, a secondary analysis was completed using PHb ≥30mg/dL. Using reference measurements, 66/88 subjects had at least one PHb level that fell within the range of detection (≥30mg/dL) of the benchtop device and 46/88 had significant hemolysis as defined by PHb ≥50mg/dL. PHb levels ≥30mg/dL largely correlated with measurements made with the benchtop device (r = 0.82, p < .001). The device was able to predict PHb values ≥30mg/dL (AUROC 0.9582) and ≥50mg/dL (AUROC 0.9637). The Bland-Altman demonstrated a mean difference of 7.0mg/dL with <5% outside the 95% limits of agreement. The HemoCue® system is an effective surrogate for more robust laboratory testing to identify clinically significant hemolysis during cardiopulmonary bypass.

Prediction of major intravascular hemolysis during pulsed electric field ablation of atrial fibrillation using a pentaspline catheter.

Pulsed electric field (PEF) has emerged as a promising energy source for catheter ablation of atrial fibrillation (AF). However, data regarding the in-vivo effect of PEF energy on erythrocytes during AF ablation procedures are scarce. This study aimed to quantify the impact of PEF energy on erythrocyte damage during AF ablation by assessing specific hemolytic biomarkers. A total of 60 patients (age: 68 years, males: 72%, serum creatinine: 91 µmol/L) with AF underwent catheter ablation of AF using PEF energy delivered by a multipolar pentaspline Farawave catheter (Farapulse, Boston Scientific, Inc.). Ablation beyond pulmonary vein isolation was performed at the operator's discretion. Peripheral venous blood was sampled for assessing the plasma levels of free hemoglobin (fHb), direct (conjugated) bilirubin, lactate dehydrogenase (LDH), and creatinine before, immediately after the ablation, and on the next day. Following the PEF ablation with duration of [median (interquartile range)] 75 (58, 95) min, with 74 (52, 92) applications and PVI only in 27% of patients, fHb, LDH, and direct bilirubin significantly increased, from 40 (18, 65) to 493 (327, 848) mg/L, from 3.1 (2.6, 3.6) to 6.8 (5.0, 7.9) µkat/L, and from 12 (9, 17) to 28 (16, 44) µmol/L, respectively (all p < .0001). A strong linear correlation was found between the peak fHb and the number of PEF applications (R = 0.81, p < .001). The major hemolysis (defined as fHb >500 mg/L) was predicted by the number of PEF applications with the corresponding area under the receiver operating characteristic curve of 0.934. The optimum cut-off value of >74 PEF applications predicted the major hemolysis with 89% sensitivity and 87% specificity. Catheter ablation of AF using PEF energy delivered from a pentaspline catheter is associated with significant intravascular hemolysis. More than 74 PEF applications frequently resulted in major hemolysis. However, the critical amount of PEF energy that may cause kidney injury in susceptible patients remains to be investigated.

Solanum dulcamara Fruit Extract: A Promising Natural Therapy for Diabetes Management?

AbstractThe study aimed to investigate the therapeutic efficacy of S. dulcamara extract (SDE) in managing type II diabetes‐related parameters, including blood glucose levels, α‐amylase activity, and modulation of endogenous antioxidant enzymes (SOD, GPx, CAT). Diabetes was induced in rats using streptozocin, and the anti‐diabetics effect was evaluated by the administration of S. dulcamara extract at different doses. Blood glucose levels, α‐amylase activity, and endogenous antioxidants were estimated and found to be significant (P&lt;0.05) in the rats treated with the extract of S. dulcamara at a dose of 200 mg/kg. The extract showed dose‐dependent inhibition of α‐amylase suggesting the potential benefits in glycemic control. Moreover, the study demonstrated a modulation in the activity of endogenous antioxidant enzymes (SOD, GPx, CAT). Importantly, the fruit extract showed no significant hemolysis or acute toxicity at varying concentrations (12.5 and 25 mg/mL) indicating the safe and biocompatible nature, as confirmed by normal biochemical parameters and histopathological examination. In conclusion, the methanolic fruit extract of S. dulcamara shows significant anti‐diabetic activity by alleviating oxidative stress associated with type II diabetes and its complications.

Screening and heterologous expression of an antimicrobial peptide SCAK33 with broad-spectrum antimicrobial activity resourced from sea cucumber proteome.

Antimicrobial peptides (AMPs) are a family of short defense proteins that are naturally produced by all organisms and have great potential as effective substitutes for small-molecule antibiotics. The present study aims to excavate AMPs from sea cucumbers and achieve their heterologous expression in prokaryotic Escherichia coli. Using MytC as a probe, a cysteine-stabilized peptide SCAK33 with broad-spectrum antimicrobial activity was discovered from the proteome of Apostichopus japonicas. The SCAK33 showed inhibitory effects on both gram positive and gram negative bacteria with MICs of 3-28μM, and without significant hemolysis activity in rat blood erythrocyte. Especially, it exhibited good antimicrobial activity against Bacillus megaterium, B. subtilis, and Vibrio parahaemolyticus with the MIC of 3, 7, and 7μM, respectively. After observation by scanning electronic microscopy (SEM) and confocal laser scanning microscope (CLSM), it was found that the cell membrane of bacteria was severely damaged. Furthermore, the recombinant SCAK33 (reSCAK33) was heterologously expressed by fusion with SUMO tag in E. coli BL21(DE3), and the protein yield reached 70mg/L. The research will supplement the existing quantity of sea cucumber AMPs and provide data support for rapid mining and biological preparation of sea cucumber AMPs.

Evaluating the biomedical and environmental safety of selenium nanoparticles synthesized from black pepper seed extract

In recent times, sustainable synthesis of selenium nanoparticles (Se NPs) has seen a significant rise in research, focusing primarily on microbial and plant polysaccharide-based approaches for various bioapplications. However, there are limited studies on the use of extracts from herbal spice plants as capping agents for Se NPs. Therefore, this study investigates the pioneering synthesis of Se NPs using black pepper (Piper nigrum) seed extract (PSe NPs) and assesses their antioxidant properties, hemolytic potential, antibacterial and antibiofilm activities, cytotoxicity, phytotoxicity, in vivo toxicity in zebrafish embryos, and their effectiveness in prolonging the shelf life of food materials. The green-synthesized PSe NPs were thoroughly physicochemically characterized and found to be sphere-shaped with a medium diameter of 10–100 nm. The PSe NPs exhibited substantial antibacterial effects, being more effective against S. aureus (Gram-positive)than against S. typhi (Gram-negative). Furthermore, PSe NPs significantly inhibited MRSA biofilm growth at 500 μg/mL and showed notable antioxidant activity compared to standard gallic acid. Also at 500 μg/mL, PSe NPs induced significant hemolysis, whereas inferior concentrations did not. Cytotoxicity evaluations using the MTT assay revealed that PSe NPs significantly reduced the viability of human normal embryonic kidney (HEK 293) cells and human cervical cancer (HeLa) cells after 24 hours of treatment at concentrations of 0.1–500 μg/mL. This cytotoxic effect was associated with apoptosis, as shown by acridine orange/ethidium bromide staining. In phytotoxicity assays, PSe NPs revealed significant effects on seed germination and root and shoot growth of the mung bean (Vigna radiata). The zebrafish embryotoxicity assay indicated that PSe NPs at 500 µg/mL affected embryo morphology, mortality and survival rates 96 hours after fertilization. Furthermore, the application of PSe NPs gel coatings effectively reduced weight loss and extended the shelf life of fruits such as apples and pears, maintaining their natural appearance.

Significant hemolysis is present during irreversible electroporation of cardiomyocytes in vitro

BackgroundPulsed field ablation (PFA) of atrial fibrillation is a new method in clinical practice. Despite a favorable safety profile of PFA in atrial fibrillation ablation, rare cases of renal failure, probably due to hemolysis, have recently been reported. ObjectiveThe aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities. MethodsBlood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation using 216 bipolar pulses, each lasting 2 μs with intervals of 5 μs, repeated 20 times at a frequency of 1 Hz. These pulses varied from 500 V to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles were evaluated by flow cytometry. Cardiomyocyte death was quantified with propidium iodide. ResultsPulsed field energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.32 ± 0.16 g/L, 2.2 ± 0.96 g/L, and 5.7 ± 0.39 g/L; P < .01) and similar increase in the concentration of red blood cell microparticles. 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 < .01). ConclusionThe 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.

Adjustment of lactate dehydrogenase concentration results according to the haemolysis index following in vitro haemolysis

In the presence of haemolysis, the interpretation of the Lactate dehydrogenase (LDH) activity result is a major operational challenge for medical laboratories: if the origin is intravascular, then the measurement will reflect the clinical reality, but in extravascular haemolysis, the laboratory will be confronted with an artefactual increase leading to false-positive high results. The aim of our study was to evaluate the adjustment of LDH concentration results according to the haemolysis index (HI). After designed a mathematical model to correct the LDH measured as a function of the haemolysis index using a Cobas 8000 analyser (Roche diagnostics, Mannheim, Germany), LDH measurement of seventy-four duplicate samples were tested before and after exposure to extravascular haemolysis process. After in vitro haemolysis process, a significant increase haemolysis index (Man-Whitney U-Test p < 0.0001) were observed. Before process the HI median was 4 [2.0 − 6.75] and after HI median was 18 [10 − 35.75]. Without correction, LDH results showed a significant increase (p < 0.001) after haemolysis process and substantial analytical discrepancies (31/74) were observed according to TEa of CLIA. After correction, data showed no significant difference (p = 0.497) and the mathematical algorithm allowed to reduce the analytical discrepancies (2/74). If haemolysis was present in vitro, the mathematical algorithm increased the accuracy of the LDH results. However, the lack of discrimination between in vivo and in vitro haemolysis requires caution and the results should be reported only as a commentary to inform the clinician.

Biomedical Applications of Green Synthesized Zinc Oxide and Magnesium-Doped Zinc Oxide Nanoparticles Using Aqueous Extract of Ziziphus Oxyphylla Leaves.

Zinc oxide (ZnO) and magnesium-doped zinc oxide (Mg-doped ZnO) nanoparticles (NPs) were synthesized using Ziziphus oxyphylla 's aqueous leaf extract as reducing agent. UV-Vis absorption peaks at 324 nm and 335 nm were indicative of ZnO and Mg-doped ZnO, respectively. FTIR absorption bands observed at 3238, 1043, 1400, 1401, 2186 and 2320 cm -1 suggested the presence of phenols, alcohols, saturated hydrocarbons, and possibly alkynes. X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy revealed pure, spherical and agglomerated NPs with average size of 35.9 nm (ZnO) and 56.8 nm (Mg-doped ZnO). Both NPs remained active against all bacterial strains with the highest inhibition zones observed against Proteus vulgaris (21.16±1.25 mm for ZnO and 24.1±0.76 mm for Mg-doped ZnO. EtBr fluorescence (cartwheel assay) indicated efflux pump blockage, suggesting its facilitation in the bacterial growth inhibition. Antioxidant potential, determined via DPPH radical scavenging assay, revealed stronger antioxidant potential for Mg-doped ZnO (IC [Formula: see text]/mL) than pure ZnO (IC [Formula: see text]/mL). Furthermore, both NPs showed antileishmanial activity against Leishmania tropica promastigotes (IC [Formula: see text]/mL for Mg-doped ZnO and 64.34±6.56 for ZnO), while neither NP exhibited significant hemolysis, indicating biocompatibility and further assessment for their drugability.

Characterizing the Racial Discrepancy in Hypoxemia Detection in Venovenous Extracorporeal Membrane Oxygenation: An Extracorporeal Life Support Organization Registry Analysis.

Skin pigmentation influences peripheral oxygen saturation (SpO2) compared to arterial saturation of oxygen (SaO2). Occult hypoxemia (SaO2 ≤ 88% with SpO2 ≥ 92%) is associated with increased in-hospital mortality in venovenous-extracorporeal membrane oxygenation (VV-ECMO) patients. We hypothesized VV-ECMO cannulation, in addition to race/ethnicity, accentuates the SpO2-SaO2 discrepancy due to significant hemolysis. Adults (≥ 18years) supported with VV-ECMO with concurrently measured SpO2 and SaO2 measurements from over 500 centers in the Extracorporeal Life Support Organization Registry (1/2018-5/2023) were included. Multivariable logistic regressions were performed to examine whether race/ethnicity was associated with occult hypoxemia in pre-ECMO and on-ECMO SpO2-SaO2 calculations. Of 13,171 VV-ECMO patients, there were 7772 (59%) White, 2114 (16%) Hispanic, 1777 (14%) Black, and 1508 (11%) Asian patients. The frequency of on-ECMO occult hypoxemia was 2.0% (N = 233). Occult hypoxemia was more common in Black and Hispanicpatients versus White patients (3.1% versus 1.7%, P < 0.001 and 2.5% versus 1.7%, P = 0.025, respectively). In multivariable logistic regression, Black patients were at higher risk of pre-ECMO occult hypoxemia versus White patients (adjusted odds ratio [aOR] = 1.55, 95% confidence interval [CI] = 1.18-2.02, P = 0.001). For on-ECMO occult hypoxemia, Black patients (aOR = 1.79, 95% CI = 1.16-2.75, P = 0.008) and Hispanic patients (aOR = 1.71, 95% CI = 1.15-2.55, P = 0.008) had higher risk versus White patients. Higher pump flow rates (aOR = 1.29, 95% CI = 1.08-1.55, P = 0.005) and on-ECMO 24-h lactate (aOR = 1.06, 95% CI = 1.03-1.10, P < 0.001) significantly increased the risk of on-ECMO occult hypoxemia. SaO2 should be carefully monitored if using SpO2 during ECMO support for Black and Hispanic patients especially for those with high pump flow and lactate values at risk for occult hypoxemia.

Resveratrol-Ampicillin Dual-Drug Loaded Polyvinylpyrrolidone/Polyvinyl Alcohol Biomimic Electrospun Nanofiber Enriched with Collagen for Efficient Burn Wound Repair.

The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries. This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair. Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31±0.09 mm and 12±0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10. The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach.

Characterization and clinical significance of hemolysis after pulsed-field ablation for atrial fibrillation: insights from a multicentric analysis

Abstract Background Pulsed-field ablation is a novel non-thermal ablation modality which is being increasingly used in clinical practice for the treatment of atrial fibrillation (AF). While the susceptibility of erythrocytes to electroporation is well-established, the effect of cardiac PFA technologies on erythrocyte destruction has not been described so far. Purpose The aim of this study was to investigate whether PFA induces clinically significant hemolysis. Methods We included 187 patients undergoing AF catheter ablation with either PFA (n=145) or RFA (n=42) at four high-volume centers. PFA was performed using a pentaspline catheter (biphasic, bipolar pulses of 2kV) and RFA with a 3.5 mm irrigated-tip catheter (40-60 W). The lesion set comprised pulmonary vein isolation (PVI) for paroxysmal AF and PVI ± additional lesions/lines for persistent AF. Established biomarkers of hemolysis, anemia and renal function were analyzed in blood samples obtained at baseline (T1), at the end of ablation (T2) and 24 hours after the procedure (T3). Results Baseline characteristics were well-balanced between groups (65.5 ± 9.6 years, 72.7% male, p&amp;gt;0.05). The ablation procedures comprised a mean of 61.4 ± 27.2 deliveries (PFA group) and a mean RF duration of 31.0 ± 16.3 min (RFA group). Atrial ablation in addition to PVI was performed in 55.2% vs. 61.9% patients, respectively (p=0.469). The direct hemolysis marker free plasma hemoglobin was significantly higher with PFA (592.8 ± 330.6 mg/dl) than with RFA (186.6 ± 215.8 mg/dl, p&amp;lt;0.001) at the end of ablation. A strong correlation was found between free plasma hemoglobin and PFA deliveries (Pearson r=0.615, p&amp;lt;0.001). PFA was further associated with a 2.2-fold increase in bilirubin (21.3 ± 11.3 vs. 9.6 ± 4.7 µmol/l), a 2.4-fold decrease in haptoglobin (0.5 ± 0.4 vs. 1.2 ± 0.4 g/l) and a 1.8-fold increase in LDH (352.7 ± 115.7 vs. 192.2 ± 51.6 U/l, T3 vs. T1, all p&amp;lt;0.001). There were no significant changes in bilirubin and haptoglobin in the RFA group (p&amp;gt;0.05). A slight decrease in red blood cells, hemoglobin and hematocrit was detected after both PFA and RFA (Abstract Figure), with no significant differences between groups (all p&amp;gt;0.05). While mean creatinine levels and glomerular filtration rates did not significantly change after ablation in either group, acute kidney injury occurred in 4/124 (3.2%) with PFA vs. 0/32 (0%) with RFA (p=0.303). Conclusion Intravascular hemolysis is a frequent finding after PFA for AF and increases with the number of PFA deliveries. While PFA was not associated with clinically significant anemia, the incidence of acute kidney injury in the present study warrants future large-scale investigations assessing the biological impact and nephrotoxicity of PFA-induced hemolysis.Abstract Figure

#2765 Medium chain fatty acids are potent binding competitors to improve protein-bound uremic toxin clearance in hemodialysis

Abstract Background and Aims Protein-bound uremic toxins (PBUTs) remain a concerning burden in patients with end-stage renal disease (ESRD) since their removal during hemodialysis is limited due to their tight binding to albumin. By increasing their circulating free fraction during HD, “binding competitors” of albumin could be used to increase their dialytic clearance. Medium chain fatty acids (MCFAs, 6 to 12 carbon length) are potent candidates for that purpose. The present study aims to investigate whether MCFAs could serve as potential binding competitors of albumin to enhance the removal of PBUTs, specifically IS and p-CS, during hemodialysis. Method Hexanoate (C6), octanoate (C8), and decanoate (C10) were purchased from Sigma-Aldrich. The binding of MCFAs to albumin was investigated in silico and using fluorescent probes: warfarin (specific of Sudlow's site I) and dansylsarcosine (specific of Sudlow's site II). Indoxyl-sulfate (IS, final concentration 212 µM) and p-cresyl sulfate (p-CS, final concentration 250 µM) were added to a 600 µM bovine serum albumin-phosphate buffered saline solution. The free fractions of IS and p-CS were assayed with or without MCFAs (1-2 mM) using ultrafiltration devices in this solutions and in patients uremic plasma. To mimic the removal of PBUTs during a HD session, batches of 2 liters of fresh bovine blood were loaded with IS and p-CS (final concentration 200 µM). A 2-hours closed-loop HD session was performed using a Fresenius 5008 CorDiax HD generator (Fresenius, Germany). A solution of 224 mM of octanoate was perfused at the rate of 150 µL/min; A solution of saline 0.9% was used as a control. One milliliter of blood was sampled every 15 minutes through the arterial sampling port and concentration of IS and p-CS were assayed by HPLC coupled with fluorescence detection. The hemolytic effect of MCFAs was evaluated in vitro by assaying the free hemoglobin concentration. Results According to the in silico prediction, the binding was predicted to be almost complete for MCFAs ≥ C8 (Fig. 1). The enhanced protein binding appeared to be inversely correlated with water solubility, which became particularly low for MCFAs ≥ C10. Octanoate and decanoate were the more prone to displace dansylsarcosine from Sudlow's site II of albumin (which is the main binding site of IS and p-CS). In vitro, the incubation with 2 mM of octanoate and decanoate increased the free fraction of PBUTs from 12% to 53% for p-CS (4.4 fold, p &amp;lt; 0.05) and from 11% to 45% for IS (4.1 fold, p &amp;lt; 0.05). Octanoate was also able to increase the free fraction of PBUTs in patients uremic plasma (1.6-fold for IS, p &amp;lt; 0.05 and 4.8 fold for p-CS, &amp;lt;0.01). The per-dialytic infusion of octanoate significantly increased the fractional removal of p-CS (from 38% to 88%, p &amp;lt; 0.001) and IS (from 36% to 91%, p &amp;lt; 0.001). No significant hemolysis was observed for concentration of MCFAs lower than 2 mmol/L. Conclusion MCFAs and especially octanoate and decanoate are serious candidates to displace the binding of PBUTs such as p-CS and IS. The per-dialytic administration of octanoate significantly increased the removal of PBUTs and could constitute a new strategy to prevent their accumulation in end-stage kidney disease patients. Due to their good safety profile, MCFAs could be better tolerated than other chemical compounds that has already been tested in a clinical setting such as ibuprofen. Further in vivo studies are however needed to carefully evaluate this potentially new therapeutic option.

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis

BackgroundDue to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication.MethodsA novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area.ResultsBiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area.ConclusionsDue to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

Periprocedural Intravascular Hemolysis During Atrial Fibrillation Ablation: A Comparison of Pulsed Field With Radiofrequency Ablation

BackgroundHemolysis-related renal failure has been described after pulmonary vein isolation (PVI) with pulsed-field ablation (PFA). ObjectivesThis study sought to compare the potential for hemolysis during PVI with PFA vs radiofrequency ablation (RFA). MethodsIn consecutive patients, PVI was performed with PFA or RFA. Blood samples were drawn at baseline, immediately postablation, and 24 hours postablation. Using flow cytometry, the concentration of red blood cell microparticles (RBCμ) (fragments of damaged erythrocytes) in blood was assessed. Lactate dehydrogenase (LDH), haptoglobin, and indirect bilirubin were measured at baseline and 24 hours. ResultsSeventy patients (age: 64.7 ± 10.2 years; 47% women; 36 [51.4%] paroxysmal atrial fibrillation) were enrolled: 47 patients were in the PFA group (22 PVI-only and 36.4 ± 5.5 PFA applications; 25 PVI-plus, 67.3 ± 12.4 pulsed field energy applications), and 23 patients underwent RFA. Compared to baseline, the RBCμ concentration increased ∼12-fold postablation and returned to baseline by 24 hours in the PFA group (median: 70.8 [Q1-Q3: 51.8-102.5] vs 846.6 [Q1-Q3: 639.2-1,215.5] vs 59.3 [Q1-Q3: 42.9-86.5] RBCμ/μL, respectively; P < 0.001); this increase was greater with PVI-plus compared to PVI-only (P = 0.007). There was also a significant, albeit substantially smaller, periprocedural increase in RBCμ with RFA (77.7 [Q1-Q3: 39.2-92.0] vs 149.6 [Q1-Q3: 106.6-180.8] vs 89.0 [Q1-Q3: 61.2-123.4] RBCμ/μL, respectively; P < 0.001). At 24 hours with PFA, the concentration of LDH and indirect bilirubin increased, whereas haptoglobin decreased significantly (all P < 0.001). In contrast, with RFA, there were only smaller changes in LDH and haptoglobin concentrations (P = 0.03) and no change in bilirubin. ConclusionsPFA was associated with significant periprocedural hemolysis. With a number of 70 PFA lesions, the likelihood of significant renal injury is uncommon.