Theories on malarial pigment formation and quinoline action

Haeme metabolism remains a vulnerable problem for the intraerythrocytic Plasmodium which catabolises haemoglobin as a source of amino acids in an acidic, oxygen-rich lysosome-like digestive vacuole. Haeme monomer, capable of generating oxygen radicals, transforms into an inert crystal named malarial pigment or haemozoin by forming unique dimers that then crystalise. Laveran first described pigmented bodies in humans to define a protozoan as the aetiologic agent of malaria. The trail of malaria pigment enabled Ross to implicate the mosquito in the life cycle of Plasmodium. In 1991, Slater and Cerami postulated a unique iron-carboxylate bond between two haemes in haemozoin crystals based on infrared and X-ray spectroscopy data. Additionally, parasite extracts were shown to possess a 'haeme polymerase' enzymatic activity as the process of crystal formation was then termed. Importantly, the quinolines, such as choloroquine, inhibit haemozoin formation. A Plasmodium falciparum derived histidine-rich protein II, which binds haeme and initiates haemozoin formation, is present in the digestive vacuole. Pfhistidine-rich protein II and Pfhistidine-rich protein III are sufficient, but not necessary for haemozoin formation as a laboratory clone lacking both still makes the haeme crystals. The reduvid bug, and the Schistosoma and Haemoproteus genera also make haemozoin. Recently, Bohle and coworkers used X-ray diffraction to document the iron-carboxylate bond in intact desiccated parasites and to show that a Fe1-O41 head to tail haeme dimer is the unit building block of haemozoin. The role of the Plasmodium histidine-rich protein family members, lipids or potential novel proteins in the exact molecular assembly of the large molecular weight haeme crystals in the protein rich digestive vacuole needs to be solved. Accurate experimental determination of the role of haemozoin formation and inhibition as the target of chloroquine is fundamental to determination of the mechanism of quinoline drug action and resistance. The enhanced understanding of the biosynthetic pathway leading to haemozoin formation using functional proteomic tools and the mechanisms through which existing antimalarial drugs affect Plasmodium haeme chemistry will help design improved chaemotherapeutic agents.     

Effect of near-infrared and blue laser light on vero E6 cells SARS-CoV-2 infection model

Photobiomodulation therapy (PBMT) employing laser light has been emerging as a safe strategy to challenge viruses. In this study the effect of blue and near-infrared (NIR) laser light was assessed in an in vitro model of SARS-CoV-2 infection. PBMT at blue wavelength inhibited viral amplification when the virus was directly irradiated and then transferred to cell culture and when cells already infected were treated. The NIR wavelength resulted less efficacious showing a minor effect on the reduction of the viral load. The cells receiving the irradiated virus or directly irradiated rescued their viability to level comparable to not treated cells. Virion integrity and antigenicity were preserved after blue and NIR irradiation, suggesting that the PBMT antiviral effect was not correlated to viral lipidic envelope disruption. Our results suggested that PBMT can be considered a valid strategy to counteract SARS-CoV-2 infection, at least in vitro.     

Photo-Irradiation of Proanthocyanidin as a New Disinfection Technique via Reactive Oxygen Species Formation

In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.     

Artemether-lumefantrine to treat malaria in pregnancy is associated with reduced placental haemozoin deposition compared to quinine in a randomized controlled trial

ABSTRACT: BACKGROUND: Data on efficacy of artemisinin-based combination therapy (ACT) to treat Plasmodium falciparum during pregnancy in sub-Saharan Africa is scarce. A recent open label, randomized controlled trial in Mbarara, Uganda demonstrated that artemether-lumefantrine (AL) is not inferior to quinine to treat uncomplicated malaria in pregnancy. Haemozoin can persist in the placenta following clearance of parasites, however there is no data whether ACT can influence the amount of haemozoin or the dynamics of haemozoin clearance. METHODS: Women attending antenatal clinics with weekly screening and positive blood smears by microscopy were eligible to participate in the trial and were followed to delivery. Placental haemozoin deposition and inflammation were assessed by histology. To determine whether AL was associated with increased haemozoin clearance, population haemozoin clearance curves were calculated based on the longitudinal data. RESULTS: Of 152 women enrolled in each arm, there were 97 and 98 placental biopsies obtained in the AL and quinine arms, respectively. AL was associated with decreased rates of moderate to high grade haemozoin deposition (13.3% versus 25.8%), which remained significant after correcting for gravidity, time of infection, re-infection, and parasitaemia. The amount of haemozoin proportionately decreased with the duration of time between treatment and delivery and this decline was greater in the AL arm. Haemozoin was not detected in one third of biopsies and the prevalence of inflammation was low, reflecting the efficacy of antenatal care with early detection and prompt treatment of malaria. CONCLUSIONS: Placental haemozoin deposition was decreased in the AL arm demonstrating a relationship between pharmacological properties of drug to treat antenatal malaria and placental pathology at delivery. Histology may be considered an informative outcome for clinical trials to evaluate malaria control in pregnancy. Trial registration REGISTRY: http://clinicaltrials.gov/ct2/show/NCT00495508.     

Continuous-flow system for large-scale ultraviolet irradiation of bacterial cells.

A quartz-flow-cell system for irradiation of large volumes of Escherichia coli cultures with ultraviolet light is described. With this system kilogram quantities of irradiated cells can be obtained for biochemical studies. Changes in respiration and in specific activities of superoxide dismutase and catalase, after an ultraviolet treatment that reduced viability of culture samples to 0.2%, were in good agreement with those for cultures irradiated (52J/m2) by a conventional small-scale method to produce the same reduction in viability.     

Continuous-flow system for large-scale ultraviolet irradiation of bacterial cells.

A quartz-flow-cell system for irradiation of large volumes of Escherichia coli cultures with ultraviolet light is described. With this system kilogram quantities of irradiated cells can be obtained for biochemical studies. Changes in respiration and in specific activities of superoxide dismutase and catalase, after an ultraviolet treatment that reduced viability of culture samples to 0.2%, were in good agreement with those for cultures irradiated (52J/m2) by a conventional small-scale method to produce the same reduction in viability.     

New water disinfection system using UVA light-emitting diodes

AIM: To evaluate the ability of high-energy ultraviolet A (UVA) light-emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation. METHODS AND RESULTS: We developed a new disinfection device equipped with high-energy UVA-LED. Inactivation of bacteria was determined by colony-forming assay. Vibrio parahaemolyticus, enteropathogenic Escherichia coli, Staphylococcus aureus and Escherichia coli DH5alpha were reduced by greater than 5-log(10) stages within 75 min at 315 J cm(-2) of UVA. Salmonella enteritidis was reduced greater than 4-log(10) stages within 160 min at 672 J cm(-2) of UVA. The formation of 8-hydroxy-2'-deoxyguanosine in UVA-LED irradiated bacteria was 2.6-fold higher than that of UVC-irradiated bacteria at the same inactivation level. Addition of mannitol, a scavenger of hydroxyl radicals (OH(*)), or catalase, an enzyme scavenging hydrogen peroxide (H(2)O(2)) to bacterial suspensions significantly suppressed disinfection effect of UVA-LED. CONCLUSION: This disinfection system has enough ability to inactivate bacteria and OH(*) and H(2)O(2) participates in the disinfection mechanism of UVA irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: We newly developed UVA irradiation system and found that UVA alone was able to disinfect the water efficiently. This will become a useful disinfection system.     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

Investigation of the Plasmodium falciparum food vacuole through inducible expression of the chloroquine resistance transporter (PfCRT)

Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein.     

Evaluation of Near-Infrared Pasteurization in Controlling Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Ready-To-Eat Sliced Ham

This study was conducted to investigate the efficacy of near-infrared (NIR) heating to reduce Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes in ready-to-eat (RTE) sliced ham compared to conventional convective heating, and the effect of NIR heating on quality was determined by measuring the color and texture change. A cocktail of three pathogens was inoculated on the exposed or protected surfaces of ham slices, followed by NIR or conventional heating at 1.8 kW. NIR heating for 50 s achieved 4.1-, 4.19-, and 3.38-log reductions in surface-inoculated S. Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively, whereas convective heating needed 180 s to attain comparable reductions for each pathogen. There were no statistically significant (P > 0.05) differences in reduction between surface- and internally inoculated pathogens at the end of NIR treatment (50 s). However, when treated with conventional convective heating, significant (P < 0.05) differences were observed at the final stages of the treatment (150 and 180 s). Color values and texture parameters of NIR-treated (50-s treatment) ham slices were not significantly (P > 0.05) different from those of nontreated samples. These results suggest that NIR heating can be applied to control internalized pathogens as well as surface-adhering pathogens in RTE sliced meats without affecting product quality.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Ultrastructural characteristics,biological activity and pharmacological relevance of synthetic malaria pigment (beta-haematin)

Malaria pigment (haemozoin, HZ) is the detoxification product of haemoglobin-derived haem of intraerythrocytic malaria parasites. At schizont rupture, haemozoin accumulates inside host phagocytic cells. The chemical structure and the spectroscopic characteristics of haemozoin are identical to those of beta-haematin (BH), a synthetic pigment obtained from Ferriprotoporphyrin IX (Fe (III) PPIX) in acidic conditions. The process of BH formation is the target of quinoline antimalarials. Here, we summarise the results of our studies on the ultrastructural characteristics, biological and pharmacological relevance of synthetic vs. native haemozoin. 1) By electron microscopy, native HZ and synthetic BH appear as dark brown crystals, morphologically indistinguishable and are internalised by phagocytes at the same extent. 2) Both HZ and BH modulate the production of cytokines (TNF and NO) and increase the susceptibility to lipid peroxidation of mouse or human phagocytes. The antioxidant status of the phagocytes regulates the susceptibility to BH/HZ-mediated effects. 3) The process of BH formation from Fe(III)PPIX, hence haem detoxification, can be inhibited by electrochemically-reduced, Fe(II)PPIX molecules. Maintaining iron in the reduced state can thus be considered a new pharmacological target. This was confirmed by the observation that thiol-reducing agents (NAC, cystein) were able to inhibit BH formation and were toxic to parasites in vitro.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater.

Giardiasis and cryptosporidiosis are diseases caused by the protozoan parasites Giardia lamblia and Cryptosporidium parvum. Waterborne transmission of these organisms has become more prevalent in recent years, and regulatory agencies are urging that source and finished water be screened for these organisms. A major problem associated with testing for these organisms is the lack of reliable methodologies and baseline information on the prevalence of these parasites in various water sources. Our study addressed both of these issues. We evaluated the presence and reduction of Giardia cysts and Cryptosporidium oocysts in sewage effluent by a combination of indirect fluorescent antibody (IFA) staining and PCR. Our results indicated a 3-log reduction of Giardia cysts and a 2-log reduction of Cryptosporidium oocysts through the sewage treatment process as determined by IFA. We developed a nested PCR to detect Cryptosporidium oocysts and used a double PCR to detect Giardia cysts. A 100% correlation was noted between IFA and PCR detection of Giardia cysts while correlation for Cryptosporidium oocysts was slightly less. On the basis of these results, PCR may be a useful tool in the environmental analysis of water samples for Giardia and Cryptosporidium organisms.     

Improvement of stored turkey semen quality as a result of He-Ne laser irradiation

Two experiments were carried out to evaluate the effects of He-Ne laser irradiation at various energy doses on the quality of stored turkey semen. Four semen pools were used in Experiment 1. Each pool was divided into 10 aliquots, nine of which were irradiated with energy doses ranging from 0.144 to 10.8 J/cm2 while the tenth one was not irradiated (control). Each sample was evaluated for motility immediately after irradiation, 24 and 48 h later. Energy doses ranging from 3.24 to 5.4 J/cm2 had higher (P <0.01) sperm motility index (SMI) value compared to the control and samples irradiated with lower and higher laser doses. The energy dose of 3.96 J/cm2 was selected for Experiment 2 to obtain further insight on its effects on turkey sperm preservation for up to 60 h. Each pool of four semen was divided into two aliquots: one represented the control and the other one was irradiated with He-Ne laser at an energy dose of 3.96 J/cm2. Each sample was evaluated for motility and viability immediately after irradiation and then at 12 h intervals up to 60 h. The cell energy charge was also measured by HPLC. Exposure to 3.96 J/cm2 increased the SMI and viability of turkey semen stored for 60 h compared to the control (P <0.05). The cell energy charge of irradiated samples was 200% higher than in the control. Laser irradiation increased the longevity of stored turkey spermatozoa, and might be a useful technique to enhance semen quality in long-term storage.     

The Plasmodium falciparum-infected red blood cell

Plasmodium falciparum, the most virulent of the human malaria parasites, causes up to one million deaths per year. The parasite spends part of its lifecycle inside the red blood cells (RBCs) of its host. As it grows it ingests the RBC cytoplasm, digesting it in an acidic vacuole. Free haem released during haemoglobin digestion is detoxified by conversion to inert crystals of haemozoin. Malaria pathology is evident during the blood stage of the infection and is exacerbated by adhesion of infected RBCs to blood vessel walls, which prevents splenic clearance of the infected cells. Cytoadherence is mediated by surface-exposed virulence proteins that bind to endothelial cell receptors. These 'adhesins' are exported to the RBC surface via an exomembrane system that is established outside the parasite in the host cell cytoplasm. Antimalarial drugs that interfere with haem detoxification, or target other parasite-specific processes, have been effective in the treatment of malaria, but their use has been dogged by the development of resistance. Similarly, efforts to develop an effective blood vaccine are hindered by the variability of surface-exposed antigens.     

Haemozoin: identification and quantification

Haemozoin (malaria pigment) is a haem polymer resulting from the breakdown of haemoglobin by Plasmodium spp. This refractory substance has been the focus of many studies and of much debate, mainly because of its role in the pharmacological activity of certain antimalarials. Haemozoin is also important because its presence in tissues serves as an indicator of malaria infections, and may itself be a mediator of malaria pathogenesis. In this article, Amy Sullivan and Steven Meshnick review the structure and synthesis of haemozoin, and then focus on methods of haemozoin identification in tissue. This latter aspect has implications for the study of haemozoin both as an indicator of malaria infection and as a possible mediator of malaria pathogenesis.     

The Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Non-sporing Bacteria

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0% (w/v) for 75 and 180 min respectively. The same kill was produced by 30 s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide.     

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.