EhVps35, a retromer component, is a key factor in secretion, motility, and tissue invasion by Entamoeba histolytica.

In humans and Drosophila melanogaster, the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation.

Red blood cell extended antigen typing in Omani patients with sickle cell disease to enhance daily transfusion practice.

Many Omani patients with sickle cell disease (SCD) undergo red blood cell (RBC) transfusions that are only matched for ABO and D, making RBC alloimmunization a significant concern in this population. Currently, the integration of molecular assays and hemagglutination testing helps to determine RBC phenotypes and genotypes, facilitating the provision of compatible blood and minimizing additional alloimmunization risks in patients with SCD. Based on this finding, our objective was to use molecular methods to predict the extended antigen profile of Omani patients with SCD across various blood group systems including Rh, Kell, Duffy, Kidd, Colton, Lutheran, Dombrock, Diego, Cartwright, and Scianna. This approach aims to implement RBC matching strategies and enhance daily transfusion practices for these patients. Molecular methods encompassed multiplex polymerase chain reaction for RHD, BeadChip arrays for variants of RHD and RHCE, and ID CORE XT for the primary allelic variants of RBCs. This study enrolled 38 patients with SCD, comprising 34 patients with homozygous HbSS, 1 patient with HbSC, and 3 patients with HbS Oman. The predominant ABO blood group was group O, observed in 44.7 percent of patients, followed by group A in 21.1 percent and group B in 13.2 percent. The most prevalent Rh phenotype predicted from the genotype was D+C+E-c+e+, identified in 34.2 percent of patients. All patient samples were K-, exhibiting the k+ Kp(b+) Js(b+) phenotype, with 81.6 percent demonstrating Fy(a-b-) due to the homozygous FY*02N.01 genotype and 28.9 percent displaying Jk(a+b-). RH variant alleles were detected in five patients (13.2 %), with only one type of RHD variant (RHD*DIIIa) and one type of RHCE variant (RHCE*ceVS.02.01) identified. Alloantibodies were present in 26 patients (68.4%). This study presents the initial comprehensive report of extended RBC antigen profiling in Omani patients with SCD, revealing disparities in the prevalence of RBC phenotypes compared with SCD patients from other regions and countries. Furthermore, our findings underscore a high rate of alloimmunization in these patients, emphasizing the need to implement antigen-matching programs to improve daily transfusion practices.

Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions.

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

Stability of immunohaematological reagents used for blood typing of recipients in the tube technique.

BACKGROUND AND OBJECTIVES: The storage temperature of immunohaematological reagents generally ranges from 2 to 8°C, and they should be utilised at room temperature. This study aimed to analyse the stability of immunohaematological reagents used in ABO and RhD typing. METHODS: The evaluation encompassed the potency, specificity, and integrity of anti-A, anti-B, anti-D, RhD control sera, and A1 and B red blood cells (RBC) reagents after long (8 h) and short (4 h) daily periods of exposure to room temperature (20-24°C), 5 days a week for 4 weeks. Additionally, the A1 and B RBC reagents were exposed daily for 11 h and 30 min at room temperature, including 30 more minutes at room temperature with simultaneous homogenisation through equipment. For the control, an aliquot of each reagent was constantly stored at refrigeration temperature, while another was exposed to room temperature for 12 h daily. Tests conducted included reaction intensity, titration, and avidity for antisera, reaction intensity, free haemoglobin determination, and electrical conductivity for the RBC reagents. RESULTS: The antisera maintained the reaction intensity. The titre and avidity of the antisera satisfied the minimum Brazilian requirements after different exposure periods. A higher free haemoglobin concentration was noted in the RBC reagents subjected to room temperature and simultaneous homogenisation, although this did not affect the potency and specificity. The electrical conductivity average of the RBC reagent remained consistent. CONCLUSION: The findings indicate that the immunohaematological reagents from a specific manufacturer are stable under the tested temperature, ensuring the quality of the results under these conditions.

Impact of nanosilver surface electronic distributions on serum protein interactions and hemocompatibility.

The translation of silver-based nanotechnology 'from bench to bedside' requires a deep understanding of the molecular aspects of its biological action, which remains controversial at low concentrations and non-spherical morphologies. Here, we present a hemocompatibility approach based on the effect of the distinctive electronic charge distribution in silver nanoparticles (nanosilver) on blood components. According to spectroscopic, volumetric, microscopic, dynamic light scattering measurements, pro-coagulant activity tests, and cellular inspection, we determine that at extremely low nanosilver concentrations (0.125-2.5µg ml-1), there is a relevant interaction effect on the serum albumin and red blood cells (RBCs). This explanation has its origin in the surface charge distribution of nanosilver particles and their electron-mediated energy transfer mechanism. Prism-shaped nanoparticles, with anisotropic charge distributions, act at the surface level, generating a compaction of the native protein molecule. In contrast, the spherical nanosilver particle, by exhibiting isotropic surface charge, generates a polar environment comparable to the solvent. Both morphologies induce aggregation at NPs/bovine serum albumin ≈ 0.044 molar ratio values without altering the coagulation cascade tests; however, the spherical-shaped nanosilver exerts a negative impact on RBCs. Overall, our results suggest that the electron distributions of nanosilver particles, even at extremely low concentrations, are a critical factor influencing the molecular structure of blood proteins' and RBCs' membranes. Isotropic forms of nanosilver should be considered with caution, as they are not always the least harmful.

Exploring the protein profile and biological activity of Crotalus molossus venom against E. coli, P. aeruginosa and S. aureus bacteria and T47D breast carcinoma cells.

Mexico has the highest diversity of snake species in the world, following Australia when considering just venomous snakes. Specifically, in Sonora, the second largest state in the country, more than 15 highly venomous species occur, including the northern black-tailed rattlesnake (Crotalus molossus). This specie's venom has not been as thoroughly researched in contrast with other Mexican vipers, nevertheless some studies report its biological activity and even pharmacological potential with antibacterial and cytotoxic activity. In this study we identified the main protein components from a pool of C. molossus venom through a gel-free proteomics approach, reporting ∼140 proteins belonging to the SVMP (38.76%), PLA2 (28.75%), CTL (11.93%), SVSP (6.03%) and LAAO (5.67%) toxin families. To study its biological activities, we evaluated its hemolytic, antibacterial, and cytotoxic activity in red blood cells, Gram positive and negative bacteria and a luminal A breast carcinoma cell line (T47D), respectively, in vitro. We report that concentrations <100 µg/mL are potentially not hemolytic and reduced the bacteria viability of E. coli and S. aureus with an IC50 of 10.27 and 11.51 µg/mL, respectively. Finally, we determined the C. molossus venom as cytotoxic against the T47D breast carcinoma cell line, with an IC50 of 1.55 µg/mL. We suggest that the evaluated cytotoxicity was due to a high abundance of SVMPs and PLA2s, since it's been reported that they affect the extracellular matrix and membrane permeation. This may provide a useful tool for pharmaceutical screening in the future.

Differences in whole blood before and after hemodialysis session of subjects with chronic kidney disease measured by Raman spectroscopy.

This study aimed to identify differences in the composition of whole blood of patients with chronic kidney disease (CKD), before and after a hemodialysis session (HDS), and possible differences in blood composition between stages and between genders using Raman spectroscopy and principal component analysis (PCA). Whole blood samples were collected from 40 patients (20 women and 20 men), before and after a HDS. Raman spectra were obtained and the spectra were evaluated by PCA and partial least squares (PLS) regression. Mean spectra and difference spectrum between the groups were calculated: stages Before and After HDS, and gender Women and Men, which had their most intense peaks identified. Stage: mean spectra and difference spectrum indicated positive peaks that could be assigned to red blood cells, hemoglobin and deoxi-hemoglobin in the group Before HDS. There was no statistically significant difference by PCA. Gender: mean spectra and difference spectrum Before HDS indicated positive peaks that could be assigned to red blood cells, hemoglobin and deoxi-hemoglobin with greater intensity in the group Women, and negative peaks to white blood cells and serum, with greater intensity in the group Men. There was statistically significant difference by PCA, which also identified the peaks assigned to white blood cells, serum and porphyrin for Women and red blood cells and amino acids (tryptophan) for Men. PLS model was able to classify the spectra of the gender with 83.7% accuracy considering the classification per patient. The Raman technique highlighted gender differences in pacients with CKD.

Irregular red blood cell antibodies, abnormal hemoglobin and dangerous universal blood donor insights from a public blood center in a Brazilian metropolitan area.

BACKGROUND: Immunohematology tests are crucial in transfusion safety. This study aimed to assess irregular red blood cell (RBC) antibodies, abnormal hemoglobin and dangerous universal blood donors at a public blood center in a Brazilian metropolitan area. METHODS: A cross-sectional study included all consecutive blood donors from January 2018 to December 2021 at the Brasília Blood Center Foundation, Federal District (FD), Brazil. RESULTS: Among 205,965 blood donations, irregular RBC antibodies were found in 743 (0.4 %). Abnormal hemoglobin was observed in 5396 (2.6 %): 3959 (1.9 %) with Hb AS, 1344 (0.7 %) with Hb AC, and 93 (< 0,1 %) with other hemoglobin variants. Of O group donors, 12.5 % (9646) had hemolysins: 12.5 % (2410) both anti-A and anti-B, 8.7 % (9646) only anti-A, and 1.6 % (1763) only anti-B hemolysins. Female sex (p < 0.001) and increasing age (p < 0.001) were associated with irregular RBC antibodies. O and/or Rh(D)-positive blood groups had a lower prevalence of irregular RBC antibodies compared to other ABO and/or Rh(D)-negative groups. Age (p < 0.001) and female sex (p < 0.001) were associated with anti-A/anti-B hemolysins, while FD residency was associated with reduced incidence (p < 0.001). CONCLUSION: Anti-A/anti-B hemolysins in O group donors, abnormal hemoglobin and irregular RBC antibodies pose risks to transfusion practice and should not be overlooked. Advancing age, female sex, ABO blood group other than O, or Rh(D)- negative are independently associated with the presence of irregular RBC antibodies. Dangerous universal blood donors were associated with advanced age, female gender, Rh(D)-positive blood type, and individuals residing in a Brazilian state other than where the blood center was located.

In vitro Effect of Cannabidiol on Red Blood Cells: Implication in Long-Lasting Pathology Treatment.

BACKGROUND: Cannabidiol (CBD) is the principal non-hallucinogenic compound of Cannabis plants with high clinical interest because CBD has been described as having anti-inflammatory, analgesic and anticonvulsant properties. CBD is considered a multitarget compound as it can interact with a wide range of targets, explaining their multiplicity of effects. Some clinical studies have indicated certain side effects of CBD, including somnolence, anemia and diarrhea, while the elevation of transaminases is considered as an exclusion criterion from the trial. Since the red blood cells (RBCs) are a source of transaminase, we assayed in vitro effect on RBCs stability. METHODS: We performed in vitro experiments with RBCs obtained from human peripheral blood with normal hematological parameters exposed to CBD in the range of therapeutic uses. We evaluated RBCs morphological changes, membrane fragility and hemoglobin release as a reflection of hemolysis. RESULTS: CBD induced an increase in the hemoglobin release (3.27 µg/106 RBC), without altered RBC osmotic fragility. When RBCs suspensions were incubated with CBD the initial number of elements (RBCs + vesicles) was increased up to 65% after 20 min and returned to basal level after 40 min of incubation. In the first 20 min, the accounts of elements were enriched in the smaller vesicles that disappeared after the remaining 20 minutes. CONCLUSION: These results suggest that CBD affects the indemnity of erythrocytes in vitro, inducing the formation of hemolytic vesicles that can provide the basis for the development of anemia, transaminase elevation and underlying tissular iron overload in patients chronically treated with CBD.

Serum Folate, Red Blood Cell Folate, and Zinc Serum Levels Are Related with Gestational Weight Gain and Offspring's Birth-Weight of Adolescent Mothers.

BACKGROUND: Gestational weight gain below or above the Institute of Medicine recommendations has been associated with adverse perinatal and neonatal outcomes. Very few studies have evaluated the association between serum and red blood cell folate concentrations and gestational weight gain in adolescents. Additionally, zinc deficiency during pregnancy has been associated with impaired immunity, prolonged labor, preterm and post-term birth, intrauterine growth restriction, low birth weight, and pregnancy-induced hypertension. OBJECTIVE: The purpose of our study is to evaluate the association between serum concentrations of zinc, serum folate, and red blood cell folate, with the increase in gestational weight and the weight and length of the newborn in a group of adolescent mothers from Mexico City. RESULTS: In our study, 406 adolescent-neonate dyads participated. The adolescents' median age was 15.8 years old. The predominant socioeconomic level was middle-low (57.8%), single (57%), 89.9% were engaged in home activities, and 41.3% completed secondary education. Excessive gestational weight gain was observed in 36.7% of cases, while insufficient gestational weight gain was noted in 38.4%. Small for gestational age infants were observed in 20.9% of the sample. Low serum folate (OR 2.1, 95% CI 1.3-3.3), decreased red blood cell folate (OR 1.6, 95% CI 1.0-2.6), and reduced serum zinc concentrations (OR 3.3, 95% CI 2.1-5.2) were associated with insufficient gestational weight gain. Decreased serum zinc levels (OR 1.2, 95% CI 1.2-3.4) were linked to an increased probability of delivering a baby who is small for their gestational age. CONCLUSIONS: Low serum folate, red blood cell folate, and serum zinc concentrations were associated with gestational weight gain and having a small gestational age baby. Both excessive and insufficient gestational weight gain, as well as having a small gestational age baby, are frequent among adolescent mothers.

Antifungal activity of Annona crassiflora Mart. dichloromethane fraction against strains of C. albicans.

Products derived from medicinal plants with antimicrobial activity are considered a promising alternative in the treatment of fungal infections. In this perspective, this study proposed to evaluate the antifungal activity of the dichloromethane fraction of Annona crassiflora Mart. against C. albicans strains. Tests were carried out to determine Minimum Inhibitory Concentration (MIC), Minimum Fungicide Concentration (MFC), microbial growth kinetics, fungal cell wall and membrane mechanisms of action, antifungal biofilm activity, and cytotoxic effects on human erythrocytes. The extract presented MIC and MFC values that ranged from 256 µg/mL to 1,024 µg/mL, with fungicidal activity in the microbial growth kinetics assay. The mechanism of action did not occur through damage to the cell wall or via binding to ergosterol in the membrane, though the fraction presents activity against biofilm and is not cytotoxic in human erythrocytes. The dichloromethane fraction of Annona crassiflora Mart. presented antifungal activity and reduced biofilm growth, without toxicity against human erythrocytes; however, further studies are needed to define its mechanism of action.

Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Anti-Leishmania effect of icetexanes from Salvia procurrens.

BACKGROUND AND PURPOSE: Leishmaniasis is a globally prevalent vector-borne disease caused by parasites of the genus Leishmania. The available chemotherapeutic drugs present problems related to efficacy, emergence of parasite resistance, toxicity and high cost, justifying the search for new drugs. Several classes of compounds have demonstrated activity against Leishmania, including icetexane-type diterpenes, previously isolated from Salvia and other Lamiaceae genera. Thus, in this study, compounds of Salvia procurrens were investigated for their leishmanicidal and immunomodulatory activities. METHODS: The exudate of S. procurrens was obtained by rapidly dipping the aerial parts in dichloromethane. The compounds were isolated by column and centrifugal planar chromatography over silica gel. The effects on L. amazonensis growth, survival, membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential and cytotoxicity of the compounds towards human erythrocytes, peripheral blood mononuclear cells and macrophages were evaluated. The effects on intracellular amastigote forms, nitric oxide (NO) and TNF-α production were also investigated. RESULTS: The exudate from the leaves afforded the novel icetexane 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2), fruticulin A (3) and demethylfruticulin A (4). The compounds (1-4) were tested against promastigotes of L. amazonensis and showed an effective inhibition of the parasite survival (IC50 = 4.08-16.26 µM). In addition, they also induced mitochondrial ROS production, plasma membrane permeability and mitochondrial dysfunction in treated parasites, and presented low cytotoxicity against macrophages. Furthermore, all diterpenes tested reduced the number of parasites inside macrophages, by mechanisms involving TNF-α, NO and ROS. CONCLUSION: The results suggest the potential of 7-hydroxyfruticulin A (1) as well as the known demethylisofruticulin A (2),fruticulin A (3) and demethylfruticulin A (4) as candidates for use in further studies on the design of anti-leishmanial drugs.

Iron incorporation in red blood cells of pediatric sickle cell anemia: a stable isotope pilot investigation.

BACKGROUND/OBJECTIVES: Sickle cell anemia (SCA) is marked by hypoxia, inflammation, and secondary iron overload (IO), which potentially modulate hepcidin, the pivotal hormone governing iron homeostasis. The aim was to evaluate the iron incorporation in red blood cells (RBC) in SCA pediatric patients, considering the presence or absence of IO. SUBJECTS/METHODS: SCA children (n = 12; SCAtotal) ingested an oral stable iron isotope (57Fe) and iron incorporation in RBC was measured after 14 days. Patients with ≥1000 ng/mL serum ferritin were considered to present IO (SCAio+; n = 4) while the others were classified as being without IO (SCAio-; n = 8). Liver iron concentration (LIC) was determined by Magnetic Resonance Imaging (MRI) T2* method. RESULTS: The SCAio+ group had lower iron incorporation (mean ± SD: 0.166 ± 0.04 mg; 3.33 ± 0.757%) than SCAio- patients (0.746 ± 0.303 mg; 14.9 ± 6.05%) (p = 0.024). Hepcidin was not different between groups. Iron incorporation was inversely associated with serum ferritin level (SCAtotal group: r = -0.775, p = 0.041; SCAio- group: r = -0.982; p = 0.018) and sickle hemoglobin (HbS) presented positive correlation with iron incorporation (r = 0.991; p = 0.009) in SCAio- group. LIC was positively associated with ferritin (SCAtotal: r = 0.921; p = 0.026) and C reactive protein (SCAio+: r = 0.999; p = 0.020). CONCLUSION: SCAio+ group had lower iron incorporation in RBC than SCAio- group, suggesting that they may not need to reduce their intake of iron-rich food, as usually recommended. Conversely, a high percentage of HbS may indirectly exacerbate hypoxia and seems to increase iron incorporation in RBC. TRIAL REGISTRATION: This trial was registered at www.ensaiosclinicos.gov.br . Identifier RBR-4b7v8pt.

Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis / Evaluación del efecto citotóxico de las semillas de Moringa peregrina en el cáncer oral, CAL 27, líneas celulares y hemólisis de glóbulos rojos

Background: Moringa peregrina Forssk is a well-known plant in ethnomedicine due to its widespread uses in various diseases like cough, wound healing, rhinitis, fever, and detoxification. The plant seeds contain compounds that are cytotoxic to many cancer cells. During the therapeutic use of plants via the oral route, some compounds present in the plants may be cytotoxic to normal cell lines and red blood cells. Objective: This study was the first report of investigation of the cytotoxic profile on oral cancer, CAL 27, cell line, and hemolytic activities on human erythrocytes of Moringa peregrina seeds ethanolic extract (MPSE). Methods: MPSE was screened for its cytotoxic effect against oral cancer, CAL 27, cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT) assay. The toxicity of MPSE on human erythrocytes was determined by in vitro hemolytic assay. Results: MPSE showed significant anti-proliferative activity against oral cancer, CAL 27 cell line at lower concentrations with half maximal inhibitory concentration (IC50) value of 21.03 µg/mL. At 1,000 µg/ml of MPSE, the maximum hemolysis was found to be 14.3% which is within safer limit. Conclusions: This study revealed a potential anti-oral cancer of MPSE and provided a baseline for its potential use in oral cancer treatment with minimum hemolytic effect on human RBCs.

La Moringa peregrina Forssk es una planta muy conocida en etnomedicina debido a sus usos generalizados en diversas enfermedades como la tos, la cicatrización de heridas, la rinitis, la fiebre y la desintoxicación. Las semillas de la planta contienen compuestos citotóxicos para muchas células cancerosas. Durante el uso terapéutico de las plantas por vía oral, algunos compuestos presentes en ellas pueden ser citotóxicos para las líneas celulares normales y los glóbulos rojos. Objetivo: Este estudio fue el primer informe de investigación del perfil citotóxico sobre el cáncer oral, CAL 27, línea celular, y las actividades hemolíticas en eritrocitos humanos del extracto etanólico de semillas de Moringa peregrina (MPSE). Métodos: Se examinó el efecto citotóxico del MPSE contra la línea celular de cáncer oral CAL 27 mediante el ensayo con bromuro de 3-(4, 5-dimetiltiazol-2-il)-2, 5,-difeniltetrazolio (MTT). La toxicidad del MPSE sobre los eritrocitos humanos se determinó mediante un ensayo hemolítico in vitro. Resultados: MPSE mostró una actividad antiproliferativa significativa contra el cáncer oral, línea celular CAL 27 a concentraciones más bajas con un valor de concentración inhibitoria media máxima (IC50) de 21,03 µg/mL. A 1.000 µg/ml de MPSE, la hemólisis máxima fue del 14,3%, lo que está dentro del límite de seguridad. Conclusiones: Este estudio reveló un potencial anticancerígeno oral de MPSE y proporcionó una base para su uso potencial en el tratamiento del cáncer oral con un efecto hemolítico mínimo en los glóbulos rojos humanos.

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.

Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Evaluating the antimicrobial and anti-biofilm activity of three synthetic antimicrobial Citropin analogs and their ability to fight against Staphylococcus aureus and Staphylococcus pseudintermedius.

AIMS: We developed three new analogs of the antimicrobial peptide (AMP) Citropin 1.1: DAN-1-13, AJP-1-1, and HHX-2-28, and tested their potential antimicrobial and antibiofilm activities against Staphylococcus aureus and S. pseudintermedius. Potential cytotoxic or hemolytic effects were determined using cultured human keratinocytes and erythrocytes to determine their safety. METHODS AND RESULTS: To assess the antimicrobial activity of each compound, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined against methicillin-resistant and methicillin-susceptible strains of S. aureus and S. pseudintermedius. Activity against newly formed and mature biofilms was determined in two clinical isolates using spectrophotometry and scanning electron microscopy (SEM). All three compounds exhibited antimicrobial and bactericidal activity against all studied S. aureus and S. pseudintermedius strains, with MICs ranging from 4-32 µg ml-1 and MBCs ranging from 8-128 µg ml-1. Subinhibitory concentrations of all compounds also showed ant-biofilm activity in the two tested isolates. All compounds exhibited limited cytotoxic and hemolytic activity. CONCLUSIONS: Novel analogs of Citropin 1.1 exhibit antimicrobial and bactericidal activities against S. aureus and S. pseudintermedius isolates and inhibit the biofilm formation of these bacteria.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.