Mechanical Characterization of the Erythrocyte Membrane Using a Capacitor-Based Technique.

Pathological processes often change the mechanical properties of cells. Increased rigidity could be a marker of cellular malfunction. Erythrocytes are a type of cell that deforms to squeeze through tiny capillaries; changes in their rigidity can dramatically affect their functionality. Furthermore, differences in the homeostatic elasticity of the cell can be used as a tool for diagnosis and even for choosing the adequate treatment for some illnesses. More accurate types of equipment needed to study biomechanical phenomena at the single-cell level are very costly and thus out of reach for many laboratories around the world. This study presents a simple and low-cost technique to study the rigidity of red blood cells (RBCs) through the application of electric fields in a hand-made microfluidic chamber that uses a capacitor principle. As RBCs are deformed with the application of voltage, cells are observed under a light microscope. From mechanical force vs. deformation data, the elastic constant of the cells is determined. The results obtained with the capacitor-based method were compared with those obtained using optical tweezers, finding good agreement. In addition, P. falciparum-infected erythrocytes were tested with the electric field applicator. Our technique provides a simple means of testing the mechanical properties of individual cells.

P. falciparum Invasion and Erythrocyte Aging.

Plasmodium parasites need to find red blood cells (RBCs) that, on the one hand, expose receptors for the pathogen ligands and, on the other hand, maintain the right geometry to facilitate merozoite attachment and entry into the red blood cell. Both characteristics change with the maturation of erythrocytes. Some Plasmodia prefer younger vs. older erythrocytes. How does the life evolution of the RBC affect the invasion of the parasite? What happens when the RBC ages? In this review, we present what is known up until now.

Microwaves can kill malaria parasites non-thermally.

Malaria, which infected more than 240 million people and killed around six hundred thousand only in 2021, has reclaimed territory after the SARS-CoV-2 pandemic. Together with parasite resistance and a not-yet-optimal vaccine, the need for new approaches has become critical. While earlier, limited, studies have suggested that malaria parasites are affected by electromagnetic energy, the outcomes of this affectation vary and there has not been a study that looks into the mechanism of action behind these responses. In this study, through development and implementation of custom applicators for in vitro experimentation, conditions were generated in which microwave energy (MW) killed more than 90% of the parasites, not by a thermal effect but via a MW energy-induced programmed cell death that does not seem to affect mammalian cell lines. Transmission electron microscopy points to the involvement of the haemozoin-containing food vacuole, which becomes destroyed; while several other experimental approaches demonstrate the involvement of calcium signaling pathways in the resulting effects of exposure to MW. Furthermore, parasites were protected from the effects of MW by calcium channel blockers calmodulin and phosphoinositol. The findings presented here offer a molecular insight into the elusive interactions of oscillating electromagnetic fields with P. falciparum, prove that they are not related to temperature, and present an alternative technology to combat this devastating disease.

Antiplasmodial activity, structure-activity relationship and studies on the action of novel benzimidazole derivatives.

Malaria cases and deaths keep being excessively high every year. Some inroads gained in the last two decades have been eroded especially due to the surge of resistance to most antimalarials. The search for new molecules that can replace the ones currently in use cannot stop. In this report, the synthesis of benzimidazole derivatives guided by structure-activity parameters is presented. Thirty-six molecules obtained are analyzed according to their activity against P. falciparum HB3 strain based on the type of substituent on rings A and B, their electron donor/withdrawing, as well as their dimension/spatial properties. There is a preference for electron donating groups on ring A, such as Me in position 5, or better, 5, 6-diMe. Ring B must be of the pyridine type such as picolinamide, other modifications are generally not favorable. Two molecules, 1 and 33 displayed antiplasmodial activity in the high nanomolar range against the chloroquine sensitive strain, with selectivity indexes above 10. Activity results of 1, 12 and 16 on a chloroquine resistance strain indicated an activity close to chloroquine for compound 1. Analysis of some of their effect on the parasites seem to suggest that 1 and 33 affect only the parasite and use a route other than interference with hemozoin biocrystallization, the route used by chloroquine and most antimalarials.

Antiplasmodial activity of Cocos nucifera leaves in Plasmodium berghei-infected mice.

Plasmodium falciparum (P. falciparum) malaria presents serious public health problems worldwide. The parasite´s resistance to antimalarial drugs has proven to be a significant hurdle in the search for effective treatments against the disease. For this reason, the study of natural products to find new antimalarials remains a crucial step in the fight against malaria. In this study, we aimed to study the in vivo performance of the decoction of C. nucifera leaves in P. berghei-infected mice. We analyzed the effectiveness of different routes of administration and the acute toxicity of the extract. Additionally, we determined the suppressive, curative and prophylactic activity of the extract. The results showed that the decoction of leaves of C. nucifera is most effective when administered intramuscularly to mice in comparison to intraperitoneal, subcutaneous and intragastric methods. We also found that organ signs of acute toxicity appear at 2000 mg/kg/day as evidenced by necropsy examination. Additionally, we found that the prophylactic effect of the extract is of 48% inhibition, however, there is no curative effect. Finally, in a 4-day suppressive assay, we found that the extract can inhibit the growth of the parasite by up to 54% at sub-toxic doses when administered intramuscularly.

Analysis of the antiparasitic and anticancer activity of the coconut palm (Cocos nucifera L. ARECACEAE) from the natural reserve of Punta Patiño, Darién.

Cocos nucifera (C. nucifera) (the coconut palm tree) has been traditionally used to fight a number of human diseases, but only a few studies have tested its components against parasites such as those that cause malaria. In this study, C. nucifera samples were collected from a private natural reserve in Punta Patiño, Darien, Panama. The husk, leaves, pulp, and milk of C. nucifera were extracted and evaluated against the parasites that cause Chagas' disease or American trypanosomiasis (Trypanosoma cruzi), leishmaniasis (Leishmania donovani) and malaria (Plasmodium falciparum), as well as against a line of breast cancer cells. While there was no activity in the rest of the tests, five and fifteen-minute aqueous decoctions of leaves showed antiplasmodial activity at 10% v/v concentration. Removal of some HPLC fractions resulted in loss of activity, pointing to the presence of synergy between the components of the decoction. Chemical molecules were separated and identified using an ultra-performance liquid chromatography (UPLC) approach coupled to tandem mass spectrometry (LC-MS/MS) using atmospheric pressure chemical ionization quadrupole-time of flight mass spectrometry (APCI-Q-TOF-MS) and molecular networking analysis, revealing the presence of compounds including polyphenol, flavone, sterol, fatty acid and chlorophyll families, among others.

Pumilacidins from the Octocoral-Associated Bacillus sp. DT001 Display Anti-Proliferative Effects in Plasmodium falciparum.

Chemical examination of the octocoral-associated Bacillus species (sp.) DT001 led to the isolation of pumilacidins A (1) and C (2). We investigated the effect of these compounds on the viability of Plasmodium falciparum and the mechanism of pumilacidin-induced death. The use of inhibitors of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) was able to prevent the effects of pumilacidins A and C. The results indicated also that pumilacidins inhibit parasite growth via mitochondrial dysfunction and decreased cytosolic Ca2+.

Volatile organic compounds associated with Plasmodium falciparum infection in vitro.

BACKGROUND: In order to identify new ways to prevent transmission of vector-borne diseases such as malaria, efforts have been made to understand how insects are attracted to humans. Vector-host interaction studies have shown that several volatile compounds play an important role in attracting mosquitoes to human targets. A headspace solid-phase micro-extraction/gas chromatography-mass spectrometry (HSPME GC-MS) analysis of the volatile organic composition of extracellular vesicles (EVs) and supernatants of ultracentrifugation (SNUs) was carried out in Plasmodium falciparum-infected cultures with high and low parasitemias. RESULTS: A list of 18 volatile organic compounds (VOCs) was obtained from the EVs of both infected and uninfected RBCs with 1,2,3-Propanetriol, diacetate (diacetin) increased in the infected EVs, regardless of the parasitemia of the culture. The supernatant analysis, however, gave off 56 VOCs, with pentane 2,2,4-trimethyl being present in all the SNUs of uninfected erythrocytes but absent from the parasite-infected ones. Standing out in this study was hexanal, a reported insect attractant, which was the only VOC present in all samples from SNUs from infected erythrocytes and absent from uninfected ones, suggesting that it originates during parasite infection. CONCLUSIONS: The hexanal compound, reportedly a low-level component found in healthy human samples such as breath and plasma, had not been found in previous analyses of P. falciparum-infected patients or cultures. This compound has been reported as an Anopheles gambiae attractant in plants. While the compound could be produced during infection by the malaria parasite in human erythrocytes, the A. gambiae attraction could be used by the parasite as a strategy for transmission.

Blood Stage Plasmodium falciparum Exhibits Biological Responses to Direct Current Electric Fields.

The development of resistance to insecticides by the vector of malaria and the increasingly faster appearance of resistance to antimalarial drugs by the parasite can dangerously hamper efforts to control and eradicate the disease. Alternative ways to treat this disease are urgently needed. Here we evaluate the in vitro effect of direct current (DC) capacitive coupling electrical stimulation on the biology and viability of Plasmodium falciparum. We designed a system that exposes infected erythrocytes to different capacitively coupled electric fields in order to evaluate their effect on P. falciparum. The effect on growth of the parasite, replication of DNA, mitochondrial membrane potential and level of reactive oxygen species after exposure to electric fields demonstrate that the parasite is biologically able to respond to stimuli from DC electric fields involving calcium signaling pathways.

Malarial hemozoin: from target to tool.

BACKGROUND: Malaria is an extremely devastating disease that continues to affect millions of people each year. A distinctive attribute of malaria infected red blood cells is the presence of malarial pigment or the so-called hemozoin. Hemozoin is a biocrystal synthesized by Plasmodium and other blood-feeding parasites to avoid the toxicity of free heme derived from the digestion of hemoglobin during invasion of the erythrocytes. SCOPE OF REVIEW: Hemozoin is involved in several aspects of the pathology of the disease as well as in important processes such as the immunogenicity elicited. It is known that the once best antimalarial drug, chloroquine, exerted its effect through interference with the process of hemozoin formation. In the present review we explore what is known about hemozoin, from hemoglobin digestion, to its final structural analysis, to its physicochemical properties, its role in the disease and notions of the possible mechanisms that could kill the parasite by disrupting the synthesis or integrity of this remarkable crystal. MAJOR CONCLUSIONS: The importance and peculiarities of this biocrystal have given researchers a cause to consider it as a target for new antimalarials and to use it through unconventional approaches for diagnostics and therapeutics against the disease. GENERAL SIGNIFICANCE: Hemozoin plays an essential role in the biology of malarial disease. Innovative ideas could use all the existing data on the unique chemical and biophysical properties of this macromolecule to come up with new ways of combating malaria.