Lysis Of Erythrocytes Research Articles

Biodiversity and Hemolytic Toxicity of the Genus Heterocapsa (Dinophyceae) in the Beibu Gulf, China

The dinoflagellate genus Heterocapsa includes several widely distributed and potentially toxic species associated with Harmful Algal Blooms (HABs), particularly affecting the Western Pacific Ocean. To reveal the biodiversity of Heterocapsa in Beibu Gulf, six strains were morphologically characterized using light and scanning electron microscopy, while large subunit rDNA (LSU rDNA) and internal transcribed spacer (ITS) were sequenced for phylogenetic analysis through maximum likelihood and Bayesian inferences. Two strains (BGERL169, BGERL170) were identified as Heterocapsa philippinensis ribotype I, three (BGERL171-BGERL173) as a new Heterocapsa philippinensis ribotype II, and one strain (BGERL174) as Heterocapsa pseudotriquetra. Cells of H. philippinensis were ovoid to spherical, yellowish-brown, with reticulate chloroplasts, and had a sausage-shaped nucleus positioned longitudinally along the dorsal side of the cell, and the theca was arranged in Po, cp, X, 5′, 3a, 7″, 6c, 5s, 5‴, 2⁗. Additionally, BGERL169 and BGERL171 showed no hemolytic toxicity in rabbit erythrocyte lysis assays. To the best of our knowledge, this research provides the first morphological and phylogenetic analysis of H. philippinensis, including the identification of a new ribotype, as well as the discovery of H. pseudotriquetra in Chinese waters. The findings contribute to the understanding of Heterocapsa species biogeography and toxicity in Chinese waters, offering valuable data for future HAB monitoring in Beibu Gulf.

Exploring time-killing and biofilm inhibition potential of bioactive proteins extracted from two varieties of Pleurotus ostreatus

IntroductionDental caries, caused by oral microbial pathogens, are a global health concern, further exacerbated by the presence of methicillin-resistant Staphylococcus aureus (MRSA). Bioactive proteins and peptides (BAPs) exhibit potent antimicrobial properties, targeting multiple cellular mechanisms within pathogens, reducing the likelihood of resistance development. Given the antimicrobial potential of BAPs, this study aimed to compare the efficacy of BAPs extracted from cultivated (Pleurotus ostreatus, PoC) and wild (Pleurotus ostreatus, PoW) mushrooms against pathogens responsible for dental caries.MethodsBAPs were extracted from both PoC and PoW using a TCA-acetone method. Antimicrobial activities were tested against seven bacteria and one fungus using agar well diffusion and MIC determination. Antibiofilm activity was assessed via modified CV assay, while DPPH and erythrocyte lysis tests evaluated free radical scavenging.ResultsPoC showed superior antimicrobial efficacy, with lower MIC and MBC values, and disrupted biofilm integrity at increasing concentrations. PoW exhibited better antioxidant activity with higher DPPH scavenging, though its antimicrobial efficacy was slightly lower than PoC.DiscussionBoth PoC and PoW BAPs inhibited dental pathogens, with PoC showing stronger inhibition against MRSA and nystatin-resistant Candida albicans. This suggests BAPs may target additional cellular mechanisms beyond membranes, PBPs, and ergosterols. Despite PoW’s stronger antioxidant properties, both BAPs had comparable antibiofilm activity. These findings suggest complementary actions of BAPs from PoC and PoW both, in treating dental caries, offering broad-spectrum antimicrobial and antioxidant benefits.

Oxidative stress and antioxidant status mong the elderly in Jakarta

Background: Indonesia is experiencing a demographic shift, with the elderly population comprising 9.92% (26.82 million) of the total population, 8.21% of whom reside in Jakarta. Oxidative stress is a key contributor to the development of degenerative diseases associated with aging, while antioxidant defenses can mitigate its effects. Objective: This study aimed to assess oxidative stress levels, measured by malondialdehyde (MDA), and antioxidant status, including catalase, reduced glutathione (GSH), and vitamin C, in the elderly population of Jakarta. Methods: A cross-sectional study was conducted with 91 elderly participants from three sub-districts in Jakarta. Erythrocyte lysate samples were analyzed to measure MDA, catalase, and GSH levels, while plasma was used to measure vitamin C. All parameters were quantified using a spectrophotometer. MDA levels and antioxidant status were categorized based on age, blood pressure, and the number of chronic diseases, with statistical analysis performed using the Kruskal-Wallis test. Results: The median MDA level was 2.68 (1.61–5.64) nmol/mL, with the highest levels observed in participants aged 60–64 and those with three chronic diseases. The mean catalase level was 2.41 ± 0.39 U/mL, the median GSH level was 6.16 (2.02–128.07) µmol/mL, and the mean vitamin C level was 10.87 ± 4.9 µg/mL. No significant differences in antioxidant status were observed based on age, blood pressure, or the number of chronic diseases. Conclusion: Oxidative stress is prevalent in the elderly and is particularly influenced by the presence of multiple chronic diseases. However, antioxidant status does not significantly vary with age, blood pressure, or disease burden in this population.

Effects of Salicylic and Acetylsalicylic Acids in Mitochondrial and Erythrocyte Membranes

For further clarification of the mechanisms of pharmacological effects of salicylic and acetylsalicylic acids, the interactions of these acids with mitochondrial and erythrocyte membranes were studied and the role of calcium ions in the effects of salicylic and acetylsalicylic acids was examined. Salicylic acid and to a lesser extent acetylsalicylic acid at 0.5−2.0 mM concentration effectively inhibited the respiratory activity of isolated rat liver mitochondria, by uncoupling respiration and phosphorylation processes, induced depolarization of the mitochondrial membrane and potentiated Ca2+-stimulated formation of mitochondrial permeability transition pores in EGTA-free media. Cyclosporine A and ruthenium red partially inhibited the mitochondrial pore opening process induced by salicylic and acetylsalicylic acids both in the absence and presence of Ca2+ ions. Salicylic acid (180–360 µM) markedly accelerated proton-induced lysis of human erythrocytes (at pH 3.2) and caused hyperpolarization of erythrocyte membranes (at pH 5.5, but not at pH 7.4), probably as a result of proton transfer to the cytoplasm of the cell. Thus, salicylic and acetylsalicylic acids interact with mitochondrial and plasma membranes, act as effective proton/Ca2+ ionophores and stimulate the mitochondrial calcium uniporter.

Hypoxia-Mimicking Mediated Macrophage-Elimination of Erythrocytes Promotes Bone Regeneration via Regulating Integrin αvβ3/Fe2+-Glycolysis-Inflammation.

Erythrocytes are the dominant component of a blood clot in terms of volume and number. However, longstanding compacted erythrocytes in blood clots form a physical barrier and make fibrin mesh more anti-fibrinolytic, thus impeding infiltration of mesenchymal stem cells. The necrosis or lysis of erythrocytes that are not removed timely can also lead to the release of pro-inflammatory toxic metabolites, interfering with bone regeneration. Proper bio-elimination of erythrocytes is essential for an undisturbed bone regeneration process. Here, hypoxia-mimicking is applied to enhance macrophage-elimination of erythrocytes. The effect of macrophage-elimination of erythrocytes on the macrophage intracellular reaction, bone regenerative microenvironment, and bone regeneration outcome is investigated. Results show that the hypoxia-mimicking agent dimethyloxalylglycine successfully enhances erythrophagocytosis by macrophages in a dose-dependent manner primarily by up-regulating the expression of integrin αvβ3. Increased phagocytosed erythrocytes then regulate macrophage intracellular Fe2+-glycolysis-inflammation, creating an improved bone regenerative microenvironment characterized by loose fibrin meshes with down-regulated local inflammatory responses in vivo, thus effectively promoting early osteogenesis and ultimate bone generation. Modulating macrophage-elimination of erythrocytes can be a promising strategy for eradicating erythrocyte-caused bone regeneration hindrance and offers a new direction for advanced biomaterial development focusing on the bio-elimination of erythrocytes.

Lysis of human erythrocytes due to Piezo1-dependent cytosolic calcium overload as a mechanism of circulatory removal

Hematopoietic stem cells surrender organelles during differentiation, leaving mature red blood cells (RBC) devoid of transcriptional machinery and mitochondria. The resultant absence of cellular repair capacity limits RBC circulatory longevity, and old cells are removed from circulation. The specific age-dependent alterations required for this apparently targeted removal of RBC, however, remain elusive. Here, we assessed the function of Piezo1, a stretch-activated transmembrane cation channel, within subpopulations of RBC isolated based on physical properties associated with aging. We subsequently investigated the potential role of Piezo1 in RBC removal, using pharmacological and mechanobiological approaches. Dense (old) RBC were separated from whole blood using differential density centrifugation. Tolerance of RBC to mechanical forces within the physiological range was assessed on single-cell and cell population levels. Expression and function of Piezo1 were investigated in separated RBC populations by monitoring accumulation of cytosolic Ca2+ and changes in cell morphology in response to pharmacological Piezo1 stimulation and in response to physical forces. Despite decreased Piezo1 activity with increasing cell age, tolerance to prolonged Piezo1 stimulation declined sharply in older RBC, precipitating lysis. Cell lysis was immediately preceded by an acute reversal of density. We propose a Piezo1-dependent mechanism by which RBC may be removed from circulation: Upon adherence of these RBC to other tissues, they are uniquely exposed to prolonged mechanical forces. The resultant sustained activation of Piezo1 leads to a net influx of Ca2+, overpowering the Ca2+-removal capacity of specifically old RBC, which leads to reversal of ion gradients, dysregulated cell hydration, and ultimately osmotic lysis.

Erythrocyte-Mimicking Nanovesicle Targeting Porphyromonas gingivalis for Periodontitis.

Porphyromonas gingivalis has been demonstrated to have the strongest association with periodontitis. Within the host, P. gingivalis relies on acquiring iron and heme through the aggregation and lysis of erythrocytes, which are important factors in the growth and virulence of P. gingivalis. Additionally, the excess obtained heme is deposited on the surface of P. gingivalis, protecting the cells from oxidative damage. Based on these biological properties of the interaction between P. gingivalis and erythrocytes, this study developed an erythrocyte membrane nanovesicle loaded with gallium porphyrins to mimic erythrocytes. The nanovesicle can target and adhere with P. gingivalis precisely, being lysed and utilized by P. gingivalis as erythrocytes. Ingested gallium porphyrin replaces iron porphyrin in P. gingivalis, causing intracellular metabolic disruption. Deposited porphyrin generates a large amount of reactive oxygen species (ROS) under blue light, causing oxidative damage, and its lethality is enhanced by bacterial metabolic disruption, synergistically killing P. gingivalis. Our results demonstrate that this strategy can target and inhibit P. gingivalis, reduce its invasion of epithelial cells, and alleviate the progression of periodontitis.

Rapid identification of bloodstream infection pathogens and drug resistance using Raman spectroscopy enhanced by convolutional neural networks.

Bloodstream infections (BSIs) are a critical medical concern, characterized by elevated morbidity, mortality, extended hospital stays, substantial healthcare costs, and diagnostic challenges. The clinical outcomes for patients with BSI can be markedly improved through the prompt identification of the causative pathogens and their susceptibility to antibiotics and antimicrobial agents. Traditional BSI diagnosis via blood culture is often hindered by its lengthy incubation period and its limitations in detecting pathogenic bacteria and their resistance profiles. Surface-enhanced Raman scattering (SERS) has recently gained prominence as a rapid and effective technique for identifying pathogenic bacteria and assessing drug resistance. This method offers molecular fingerprinting with benefits such as rapidity, sensitivity, and non-destructiveness. The objective of this study was to integrate deep learning (DL) with SERS for the rapid identification of common pathogens and their resistance to drugs in BSIs. To assess the feasibility of combining DL with SERS for direct detection, erythrocyte lysis and differential centrifugation were employed to isolate bacteria from blood samples with positive blood cultures. A total of 12,046 and 11,968 SERS spectra were collected from the two methods using Raman spectroscopy and subsequently analyzed using DL algorithms. The findings reveal that convolutional neural networks (CNNs) exhibit considerable potential in identifying prevalent pathogens and their drug-resistant strains. The differential centrifugation technique outperformed erythrocyte lysis in bacterial isolation from blood, achieving a detection accuracy of 98.68% for pathogenic bacteria and an impressive 99.85% accuracy in identifying carbapenem-resistant Klebsiella pneumoniae. In summary, this research successfully developed an innovative approach by combining DL with SERS for the swift identification of pathogenic bacteria and their drug resistance in BSIs. This novel method holds the promise of significantly improving patient prognoses and optimizing healthcare efficiency. Its potential impact could be profound, potentially transforming the diagnostic and therapeutic landscape of BSIs.

Haemotrophic Mycoplasmas Infecting Pigs: A Review of the Current Knowledge.

Haemotrophic mycoplasmas (haemoplasmas) are a group of highly specific and adapted bacteria. Three different haemoplasma species in pigs are known to date: Mycoplasma (M.) suis, M. parvum and 'Candidatus (Ca.) M. haemosuis'. Even though these bacteria have been known in pig farming for a long time, it is difficult to draw general conclusions about the relevance of their infections in pigs. This review summarizes the current knowledge on the three porcine haemoplasma species with regards to clinical and pathological descriptions, pathobiology, epidemiology and diagnostics as well as prevention and therapy. Overall, it is clear that considerably more data are available for M. suis than for the other two species, but generally, porcine haemoplasmas were found to be highly prevalent all over the world. Mycoplasma suis is the most virulent species, causing acute infectious anaemia in pigs (IAP), whereas M. parvum usually results in chronic and subclinical infections associated with performance losses. Little is known about the clinical significance of the recently discovered third porcine species 'Ca. M. haemosuis'. So far, the described pathogenic mechanisms mainly include direct destruction of erythrocytes via adhesion, invasion, eryptosis and nutrient scavenging, indirect erythrocyte lysis due to immune-mediated events and immune dysregulation processes. A review of published diagnostic data confirms PCR assays as the current standard method, with various cross-species and species-specific protocols. Overall, there is a need for further examination to obtain valuable insights for practical application, specifically regarding the importance of subclinical infections in naturally infected animals. An essential requirement for this will be to gain a more comprehensive understanding of the mechanisms operating between the host and the pathogen.

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

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

Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

MICROWAVE AIDED SYNTHESIS OF ZINC OXIDE NANOCERAMICS: A POTENT BIOACTIVE AGENTS WITH THERAPEUTIC CHARACTERS

Nanoparticles have been inexhaustibly utilized in Nanochemistry cohere with Bio-Technology to upgrade the immobilization and movement of catalysts in pharmaceutical nanoengineering for conveyance of helpful specialists in incessant illness diagnostics and in sensors.Herein communication we report microwave irradiation technique implemented for the preparation of novel Zinc oxide nanoparticles with beneficial bio-efficacy wherein the method employed has the advantages of producing small particle size metal oxide with high purity owing to short reaction time. We successfully synthesized and characterized two Zinc oxide based Cs2 doped ZnO2 and Li2 doped ZnO2 nanoparticles and were subjected to in vitro free radical scavenging assay wherein all the particles could scavenge DPPH and Nitric oxide radicals with less IC50 value over the positive control. Microbicidal properties of nanoparticles were proved by its effectiveness against both Gram positive and Gram negative bacteria tested besides its action against Candida albicans. Both nanoparticles synthesized showed a prominent inhibition of erythrocyte lysis by PhospholipaseA2 in a concentration dependent manner indicating better anti-inflammatory agent with possibility of preventing cancer. In this study we demonstrate by shell less CAM assay that synthesized nanoparticles could inhibit in vivo angiogenesis which would otherwise under abnormal conditions causes the acceleration of several inflammatory diseases in spite of its role in normal growth and wound healing process. Inhibition of such process is the promising methodology to hinder the progression of diseases. Present characteristic features make use of ZnO2 based doped nanoparticles to be a possible approach as a therapeutic molecule with favourable biological performance.

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

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

Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi (Lates calcarifer)

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections lead to an inflammatory response in the host that is characterized by changes in leukocyte profile and activity. We hypothesized that the profile of circulating peripheral blood leukocytes and markers of their activity, such as reactive oxygen species production, would change following an event of bacterial infection in fish. We therefore developed a flow cytometry-based methodology for analyzing blood leukocytes in barramundi (Lates calcarifer), then used it to compare healthy fish to those infected with a common pathogenic bacterium, Vibrio harveyi. The initial step of the analysis relied on the effective separation of erythrocytes and leukocytes, and two methods were tested: one based on hypotonic lysis of red blood cells, and the other involving stain-based differentiation using carboxyfluorescein succinimidyl ester (CFSE). Fluorescence-activated cell sorting analysis revealed significant differences in leukocyte parameters of infected and control fish using the stain-based approach vs. hypotonic lysis. Changes in blood leukocytes included an increase in the proportion of monocytes and decrease in that of lymphocytes in infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time in infected fish as compared to controls. Monitoring peripheral blood leukocytes provides a non-lethal analysis of inflammation that allows repeated sampling of individual fish over time. This is beneficial given the high variability between individual fish. The use of CFSE-staining for leukocyte isolation proved to be advantageous over a method based on lysis of erythrocytes.

Dual therapeutic approach: Biodegradable nanofiber scaffolds of silk fibroin and collagen combined with silver and gold nanoparticles for enhanced bacterial infections treatment and accelerated wound healing

The development of novel multifunctional wound healing materials based on natural bioactives has garnered significant interest in recent years due to their ability to stimulate the healing process and alleviate problems associated with standard wound dressings. In the presented work, a bimetallic combination of silver (Ag) and gold (Au) nanoparticles incorporated into silk fibroin (SF) and collagen (CL) composite nanofiber (CNF) using the electrospinning technique. The physicochemical investigations confirmed the successful integration of Ag and Au nanoparticles sizes of approximately 6.76 nm and 9.75 nm, with an average nanofiber diameter of 189.46 ± 108.73 nm. The evaluation of composite nanofiber treated in the in vitro antibacterial tests zone of inhibition value S. aureus (17 mm) and E. coli (18 mm). Additionally, we analyzed the cell proliferation study using NIH3T3 fibroblast cell line treated with the composite nanofiber material. The SF/CL/Ag–Au composite nanofiber exhibited the highest level of cell proliferation compared to other matrices. This increased cell proliferation offers significant advantages in wound healing applications. Furthermore, in vivo study on wound healing activity in Sprague-Dawley rats demonstrated a remarkable healing percentage of 98.85%. These findings collectively highlight the promising potential of the SF/CL/Ag–Au composite nanofiber in promoting accelerated wound healing. The composite nanofibers demonstrated remarkable hemocompatibility of erythrocyte lysis percentages varying between 1.5% and 3.9%. Besides, swelling tests revealed that the material can absorb exudates from wound sites, facilitating tissue regeneration and cell adhesion. Finally, this research highlights the potential of the SF/CL/Ag–Au composite nanofiber excellent antibacterial activity, highest biocompatibility, enhanced wound healing, and highly suitable for various biomedical applications.

Abstract 2715: Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors

Abstract Background: Innate immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), dominate the immune landscape within "cold" solid tumors, which typically exhibit poor responses to immune checkpoint inhibitors (ICIs). The dearth of tumor-infiltrating T cells and the prevailing immunosuppressive microenvironment contribute to this ineffectiveness. To bolster immunotherapy against such tumors, we developed SM2248, a bispecific VHH antibody. SM2248 simultaneously obstructs the CD47-SIRPα phagocytosis checkpoint pathway and conditionally activates CD40 on tumor-infiltrating innate immune cells. CD47/SIRPa blockade enhances macrophage and dendritic cell (DC) phagocytosis and cross-presentation of antigens to cytotoxic T cells. CD40 activation fosters DC licensing, reprograms macrophages, and guides MDSC differentiation towards DCs and macrophages. The combined action of CD47 blockade and CD40 stimulation remodels the tumor microenvironment, fostering T cells infiltration and activation. Methods: SM2248 was developed by fusing a highly selective CD47-blocking VHH module - minimizing binding to human erythrocytes and platelets, with a conditional CD40 agonistic VHH module - selectively activating immune cells upon CD47 engagement, incorporated into an inactivated IgG1 Fc backbone. The specificity and affinity of SM2248 binding to human CD47 and CD40 were assessed via ELISA and flow cytometry assays. CD47 and SIRPα interaction blockade was confirmed through a competitive ligand binding assay. Target-dependent CD40 activation was evaluated via a CD40-NFκB-Luc reporter assay. In vivo anti-tumor efficacy was determined using a syngeneic transplantation model of MC38-hCD47 cancer cells in B-hCD40 humanized mice. Binding to erythrocytes and platelets, compared with magrolimab, was assessed using flow cytometry. Hemolysis toxicity was evaluated through an in vitro erythrocyte lysis assay. Results: SM2248 effectively inhibits cell surface CD47-SIRPα interaction at nanomolar concentrations and activates CD40-NF-κB-luciferase reporters at picomolar concentrations in a target-dependent manner. In contrast to the CD47 monoclonal antibody magrolimab, SM2248 does not bind to human erythrocytes or platelets and does not induce hemolysis. In murine syngeneic tumor models, SM2248 completely halts the growth of MC38-hCD47 tumors in humanized B-hCD40 mice and does not induce dose-dependent hepatotoxicity at doses up to 10 mg/kg. Conclusions: SM2248, a humanized bispecific tetravalent VHH antibody targeting CD47 and CD40, exhibits promising in vivo efficacy and a favorable safety profile in preclinical studies, offering the potential for enhancing immunotherapy in solid tumors. Citation Format: Xiaodan Liu, Hong Zhou, Shihao Lu, Huiqin Geng, Wenjing Song, Siyao Xie, Yi Wei, Xing Zhang, Simin Yang, Sheila Zhou, Yanbin Liang. Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2715.

Effect of folic acid on isoprenaline-induced myocardial injury in rats.

Isoprenaline (ISO), a synthetic catecholamine and a β-adrenoceptor agonist, is widely used to develop an experimental model of myocardial injury (MI) in rats. The leading hypothesis for ISO-induced MI in rats is that it results from catecholamine overstimulation, oxidative stress, inflammatory responses, and development of cardiomyopathy during ISO administration. Folic acid (FA) reduces oxidative stress, improves endothelial function and prevents apoptosis, thereby contributing to cardiovascular protection. This study aimed to investigate the potentially protective effect of FA pretreatment on ISO-induced MI in rats. For 7 days, adult male Wistar albino rats were pretreated with 5 mg/kg/day of FA. On the sixth and seventh days, MI in rats was induced by administering 85 mg/kg/day of ISO. Prooxidant markers in plasma samples, antioxidant capacity in erythrocyte lysates, cardiac damage markers, lipid profile, electrocardiography (ECG) and histopathological analysis were evaluated. FA pretreatment significantly alleviated changes induced by ISO; it decreased the homocysteine and high-sensitivity troponin I level. FA moderately decreased the reactive oxygen species (ROS) levels (superoxide anion radical, hydrogen peroxide and thiobarbituric acid reactive substances) and improved the antioxidant activities of catalase, superoxide dismutase and reduced glutathione. ISO reduced the nitrite level and FA significantly alleviated this change. It can be concluded that FA, as a mild antioxidant, could be an appropriate cardioprotective substance in the rat model of ISO-induced MI.

Development of a Novel Peptide with RGD Tumor Homing Motif: Evaluation of its Anticancer Potential in Hepatocellular Carcinoma and Colon Cancer Cells

Background: Peptide-based therapy has emerged as a promising avenue for treating various disorders, and recent research has highlighted the potential of anti-cancer peptides (ACPs) in cancer treatment. In this context, this study aimed to design a novel peptide incorporating a tumor-homing peptide (RGD) and C-amidation to enhance its anticancer activity, particularly against liver (HepG2) and colon (HCT-116) cancer cell lines. Objectives: The primary objective was to design a peptide with improved anticancer properties by leveraging the tumor-homing capabilities of RGD and enhancing its activity through C-amidation. The study sought to evaluate the cytotoxicity of the designed peptide against red blood cells (RBCs) and normal Vero cells. Furthermore, the anticancer efficacy of the peptide was assessed in hepatocellular carcinoma (HepG2) and colon cancer (HCT-116) cell lines. The specific objectives included examining the apoptotic induction and morphological changes in treated cells compared to untreated cells. Methods: The peptide was designed using the ACPred-FL bioinformatics tool, and its cytotoxicity was assessed through hemolysis assays against RBCs and normal Vero cells. Anticancer activity was evaluated against HepG2 and HCT-116 cell lines. The analysis of apoptotic induction involved measuring the relative gene expression of oncogenic marker BCL2 and apoptotic markers (BAX, BID, CAS-8). Additionally, Cytopathological examination and Western Blot analysis were employed to study morphological changes and confirm the quantification of relevant markers. Results: The designed peptide, consisting of twelve amino acids with a molecular mass of 1230.6233 Da and an isoelectric point of 9.81, exhibited low erythrocyte lysis and minimal toxicity to normal cells. The IC50 values demonstrated significant anticancer activity against both HepG2 (36.49±2.6 μg/mL) and HCT-116 (11.03±2.5 μg/mL) cell lines. Treated cells exhibited a significant decrease in the oncogenic marker BCL2 and an upregulation of apoptotic markers (BAX, BID, CAS-8). Western Blot analysis confirmed these findings, and Cytopathological examination revealed scattered apoptotic and degenerative changes. Conclusion: The designed peptide displayed remarkable anticancer activity against hepatocellular carcinoma and colon cancer cell lines, effectively modulating apoptotic and oncogenic markers. These findings highlight the potential of the peptide as a therapeutic agent for cancer treatment, emphasizing its clinical significance in combating liver and colon cancers. Nonetheless, further research and development are warranted to explore the translational potential of this peptide in clinical settings.

Ligation of cervical lymphatic vessels decelerates blood clearance and worsens outcomes after experimental subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) is characterized by the extravasation of blood into the subarachnoid space, in which erythrocyte lysis is the primary contributor to cell death and brain injuries. New evidence has indicated that meningeal lymphatic vessels (mLVs) are essential in guiding fluid and macromolecular waste from cerebrospinal fluid (CSF) into deep cervical lymph nodes (dCLNs). However, the role of mLVs in clearing erythrocytes after SAH has not been completely elucidated. Hence, we conducted a cross-species study. Autologous blood was injected into the subarachnoid space of rabbits and rats to induce SAH. Erythrocytes in the CSF were measured with/without deep cervical lymph vessels (dCLVs) ligation. Additionally, prior to inducing SAH, we administered rats with vascular endothelial growth factor C (VEGF-C), which is essential for meningeal lymphangiogenesis and maintaining integrity and survival of lymphatic vessels. The results showed that the blood clearance rate was significantly lower after dCLVs ligation in both the rat and rabbit models. DCLVs ligation aggravated neuroinflammation, neuronal damage, brain edema, and behavioral impairment after SAH. Conversely, the treatment of VEGF-C enhanced meningeal lymphatic drainage of erythrocytes and improved outcomes in SAH. In summary, our research highlights the indispensable role of the meningeal lymphatic pathway in the clearance of blood and mediating consequences after SAH.

Chemico-pharmacological evaluation of the methanolic leaf extract of Catharanthus ovalis: GC-MS/MS, in vivo, in vitro, and in silico approaches.

Introduction: Natural plant-based medicines have gained popularity recently as a major source of inventive, risk-free, and more potent secondary bioactive compounds with medicinal potential. Catharanthus ovalis is a perennial shrub containing various indole alkaloids cultivated extensively for local medical purposes. Methods: This research is conducted to identify the phytocompounds present in the leaves of C. ovalis and its central and peripheral analgesic, thrombolytic, and membrane-stabilizing activities through tail immersion, acetic acid-induced writhing, human blood clot lysis, and erythrocyte lysis by heat and hypotonic solution methods, respectively. Results and discussion: A total of 39 compounds were identified using GC-MS/MS techniques, including hexadecanoic acid, methyl ester (56.749%), methyl stearate (29.782%), carvacrol and its TBDMS derivative (12.586%), and 9-octadecenoic acid, methyl ester, (E)-] (9.297%) presented in high quantity. The highest tail immersion latency was observed for the 600mg/kg extract of C. ovalis crude extract. Both 400 and 600mg/kg doses of C. ovalis crude extract exhibited prominent peripheral analgesic activity. The maximum thrombolytic effect was observed by DCM soluble fraction extract by inhibiting 54.87% of the clot. However, the aqueous-soluble fraction of this extract manifested an excellent membrane-stabilizing effect by showing 73.98% and 87.51% hemolysis against heat- and hypotonic-induced hemolysis, respectively. Some of the compounds were identified as active agents against different receptors related to these diseases, which supported the findings of in vitro and in vivo tests. Conclusion: Further investigation needs to be conducted to specify and identify the exact mechanism of action of these compounds.