Hemozoin is a potential threat in cerebral malaria pathology through the induction of RBC-EC cytoadherence.

Cerebral malaria is an outcome of multifaceted and complicated condition. Cytoadherence is one critical factor in cerebral malaria pathology as high order cytoadherence complexes result in vascular congestion and cell apoptosis. Morphological abnormalities in uninfected RBCs can be a contributing factor to aggravate cytoadherence. Malaria pigment hemozoin is a potential bioactive molecule and the role of this pigment in cerebral malaria pathology is not completely understood. To understand this, primarily we investigated the impact of hemozoin pigment on uninfected RBCs. Secondarily, we investigated the role of this pigment in formation of endothelial cells-RBCs (EC-RBC) cytoadherence complex. We first observed that a dose dependent hemozoin exposure to uninfected RBCs induced structural abnormalities. Differential counting of these abnormal RBCs indicated population of acanthocytes, spherocytes and microcytes. The formation of abnormal RBCs was observed with phosphatidylserine externalization. Lipid peroxidation, reduced glutathione and reactive oxygen species (ROS) levels indicated an increase in hemozoin exposure mediated oxidative stress. Our in-vitro cytoadherence assay indicated formation of endothelial EC-RBC cytoadherence complex. The dose dependent hemozoin exposure to uninfected RBCs resulted in oxidative stress mediated high order cytoadherence complex formation. This effect was reversed in presence of antioxidant molecules. The inhibitory effect of antioxidant molecules indicates that oxidative stress can be a regulatory factor to control cerebral malaria pathology. Being the first report to highlight the impact of malaria pigment hemozoin on uninfected RBCs, this study brings attention to the role of abnormal RBCs in worsening of cerebral malaria pathology.

An overview of biochemically characterized drug targets in metabolic pathways of Leishmania parasite.

Leishmaniasis is a neglected tropical disease with no effective vaccines to date. Globally, it affects around 14 million people living in undeveloped and developing countries. Leishmania, which is the causative eukaryotic organism, possesses unique enzymes and pathways that deviates from its mammalian hosts. The control strategy against leishmaniasis currently depends on chemotherapeutic methods. But these chemotherapeutic therapies possess several side effects, and therefore, the identification of potential drug targets has become very crucial. Identification of suitable drug targets is necessary to design specific inhibitors that can target and control the parasite. These unique enzymes can be used as possible drug targets after biochemical characterization and understanding the role of these enzymes. In this review, the authors discuss various metabolic pathways that are essential for the survival of the parasite and can be exploited as potential drug targets against leishmaniasis.

Insulin signalling in RBC is responsible for growth stimulation of malaria parasite in diabetes patients.

A cross-talk between diabetes and malaria within-host is well established. Diabetes is associated with modulation of the immune system, impairment of the healing process and to disturb the host metabolism to contribute towards propagation of parasite infection. Glucose metabolism in host is maintained by insulin and RBC has 2000 insulin receptor present on plasma membrane. These receptors are robust to relay down-stream signaling in RBCs but role of intracellular signaling in parasite growth is not been explored. The malaria parasite treated with insulin (100 ng/ml) is giving stimulation in parasite growth. The effect is lasting for several generations resulting into high parasitemia. Insulin signaling is phosphorylating protein in infected RBCs and level is high in parasite RBCs compared to uninfected RBCs. It is phosphorylating Spectrin-(α/ß), Band-4.2, Ankyrin and the other proteins of RBC cytoskeleton. It in-turn induces enhanced glucose uptake inside infected RBCs. There is a high level of infection of normal RBCs by merozoites. In summary, insulin and glucose metabolism plays a crucial role in parasite propagation, disease severity and need consideration while treating patients.

Severe malaria: Biology, clinical manifestation, pathogenesis and consequences.

Every year, millions of people are infected with malaria, resulting in significant economic losses to the developing and developed nations. The malaria parasite pursues a complicated life cycle in an invertebrate, mosquito and vertebrate host with several distinct stages. In the human host, it invades the liver and red blood cells to complete its life cycle. It is surprising that not only these two organs are under pressure and exhibit functional abnormalities; a large number of clinical studies also support the notion that malaria parasite propagation in the host affects several other organs and modulates functional outcomes of individual cells. Moreover, patients recovered from severe malaria may suffer throughout their life from impairments in organ function such as loss of eyesight, kidney failure, and much more. Thus, malaria infection leads to several pathological outcomes involving different organs and individual cells in the host. The sole purpose of the present article was to give an overview of pathological outcomes during severe malaria along with their molecular mechanisms. A large proportion of deaths associated with disease is contributed by the pathological effect in host due to parasite propagation and toxicity of antimalarials or combination of both. Hence, there is a need, not only to develop antiparasitic agents but also to discover lead molecules to take care of pathophysiological effects in the host. This may help a beginner to get involved with the topic and initiate research work towards improving adjuvant therapy or avoiding serious complications.

Plasmodium falciparum PFI1625c offers an opportunity to design potent anti-malarials: Biochemical characterization and testing potentials in drug discovery.

Putative PFI1625c was cloned, over-expressed and purified to homogeneity. It is a 56.2 kDa monomeric protease which preferentially catalyzes the degradation of gelatin with a Km = 30µM. It is a slow acting enzyme with optimal pH 8.5 and temperature 37 °C, and activity is sensitive to metalloprotease inhibitor 1,10-phenanthroline. PFI1625c active site was probed with a series of heterocyclic compounds and three molecules namely, BNPC-Inh2, DDBM-Inh1 and BHPM-Inh1 from the series were inhibiting PFI1625c protease activity. These heterocyclic compounds were found to irreversible inhibiting PFI1625c protease activity. Parasite culture was treated with these inhibitors and PFI1625c isolated from culture was found to be inactive without affecting other gelatinases present in the parasite. These inhibitors were used to generate chemically knockout PFI1625c in the parasite. PFI1625c knockout parasite remained at ring stage and was unable to complete its erythrocytic schizogony. Also, these knockout parasites were incapable to multiply. More careful analysis indicate these parasites develop oxidative stress as evident by the increase in lipid peroxidation, protein-carbonyl and a decrease of GSH level. In summary, the current study has employed biochemical, computational and pharmacological approaches to explore the role of PFI1625c in the parasite, its utility as a potential drug target to develop anti-malarials.

Remarkably Efficient Blood-Brain Barrier Crossing Polyfluorene-Chitosan Nanoparticle Selectively Tweaks Amyloid Oligomer in Cerebrospinal Fluid and Aß1-40.

Amyloid oligomers have emerged as a key neurotoxin in Alzheimer's dementia. Amyloid aggregation inhibitors and modulators have therefore offered potential applications in therapeutics and diagnosis. However, crossing the blood-brain barrier (BBB) and finding the toxic aggregates among aggregates of different sizes and shapes remain a challenge. The ability of identifying early aggregates can provide a new approach to find inhibitors of the initial nucleation events correlating presenile dementia. In this study, we have prepared polyfluorene nanoparticles using chitosan as an additive, which enables it to cross BBB efficiently and employed as a highly efficient amyloid oligomer modulator. The polymer conjugate, polyfluorene-chitosan (PC), shows no toxicity in MTT assay and precludes self-aggregation of Aß1-40 and human cerebrospinal fluid oligomers to final fibril formation. This modulation strategy is supported by thioflavin T assay, circular dichroism studies, atomic force microscope images, and Fourier transform infrared analysis. The polymer-protein interface exhibits the presence of co-aggregates and responded with a stable optical response. The simple synthesis to get desired sizes and shapes with necessary photophysical behavior, biocompatibility, and most prominently BBB permeability makes this polymer conjugate very unique and highly attractive for modulation of amyloid oligomers selectively as well as for developing next generation nanotheranostic materials toward presenile dementia.

Modulating Early Stage Amyloid Aggregates by Dipeptide-Linked Perylenebisimides: Structure-Activity Relationship, Inhibition of Fibril Formation in Human CSF and Aß1-40.

Amyloid aggregation is observed in many neurodegenerative diseases, but the formation of final plaque seldom correlates to the disease severity. Early and intermediate structures such as soluble oligomers are considered as primary toxic species in protein misfolding diseases specifically linked to Aß in Alzheimer's disease (AD). Two peptide-linked perylenebisimide isomers (PAPAP and APPPA) were developed to study the structure-activity relationship with a toxic Aß oligomer in commercial Aß as well as in human cerebrospinal fluid (CSF), diminish and inhibit them, and prevent them from forming toxic amyloid fibrils from an early stage. Self-aggregation of perylenebisimides enables the formation of nano/micro-objects that are used to interact with the hydrophobic regions of the peptide and direct the peptide aggregation into an "off-pathway", preventing mature fibril formation. Remarkably, one of the Ala-Phe dipeptide-linked perylenebisimide isomers (APPPA) showed a high selectivity toward an Aß oligomer and could also cross the endothelial monolayer barrier (blood-brain barrier, BBB) more efficiently than the other derivative (PAPAP). Kinetic ThT studies and AFM imaging provided strong proof of both of the isomers being able to inhibit fibrillation of prefibrillar and oligomeric Aß in both the commercial Aß1-40 peptide as well as in the real human CSF sample. Further, a correlation has been built using pristine fluorescence of perylenebisimides, showing modulation and "oligo-blocking". The obtained data provides clear evidence that the mutual aggregation between the modulator and amyloid aggregate becomes predominant compared to their individual aggregation. These results reinforce the development of the structural platform design to diminish toxic oligomers, inhibit them, and prevent the formation of toxic amyloid fibrils at an early stage.

Cloning, Overexpression, Purification and Immunolocal-ization of PFD0975w from the Malaria Parasite Plasmodium falciparum.

Malaria is a parasitic disease, widespread along the tropical regions of the world. The disease has killed 4, 38,000 individuals in the year 2015 (WHO). The malaria parasite, Plasmodium falciparum, has evolved resistance to front-line antimalarials over the decade, necessitating the identification of new drug targets. Protein kinases are excellent drug targets since they participate in critical cell-signaling cascades. We have identified a putative RIO-like protein kinase, PFD0975w, from the Plasmodium kinome. It is believed to play a key role in ribosome biogenesis. We have cloned and over-expressed the protein in E. coli and purified it to homogeneity. The recombinant protein is of molecular weight 36.3±1.2 kDa. Purified recombinant PFD0975w is active in vitro and binds ATP. PFD0975w exhibits a unique localization pattern in each RBC stage. PFD0975w localizes within the parasite cytosol during ring stage and spread throughout the infected RBCs during trophozoite and schizont stages with the strongest expression signal during the trophozoite phase indicating the importance of the enzyme in parasite growth and survival. Interestingly, the localization pattern of the protein also responds to stress conditions such as starvation and antimalarial drug pressure. It exhibits punctuate pattern in the treated parasite during trophozoite and schizont stages compared to untreated parasites, indicating some role of the putative kinase in cellular stress handling. Our results indicate PFD0975w is a potential drug target in the malaria parasite and active recombinant PFD0975w can be exploited to identify, validate or design novel inhibitors.

Multiple function fluorescein probe performs metal chelation, disaggregation, and modulation of aggregated Aß and Aß-Cu complex.

An exceptional probe comprising indole-3-carboxaldehyde fluorescein hydrazone (FI) performs multiple tasks, namely, disaggregating amyloid ß (Aß) aggregates in different biomarker environments such as cerebrospinal fluid (CSF), Aß1-40 fibrils, ß-amyloid lysozyme aggregates (LA), and U87 MG human astrocyte cells. Additionally, the probe FI binds with Cu(2+) ions selectively, disrupts the Aß aggregates that vary from few nanometers to micrometers, and prevents their reaggregation, thereby performing disaggregation and modulation of amyloid-ß in the presence as well as absence of Cu(2+) ion. The excellent selectivity of probe FI for Cu(2+) was effectively utilized to modulate the assembly of metal-induced Aß aggregates by metal chelation with the "turn-on" fluorescence via spirolactam ring opening of FI as well as the metal-free Aß fibrils by noncovalent interactions. These results confirm that FI has exceptional ability to perform multifaceted tasks such as metal chelation in intracellular conditions using Aß lysozyme aggregates in cellular environments by the disruption of ß-sheet rich Aß fibrils into disaggregated forms. Subsequently, it was confirmed that FI had the ability to cross the blood-brain barrier and it also modulated the metal induced Aß fibrils in cellular environments by "turn-on" fluorescence, which are the most vital properties of a probe or a therapeutic agent. Furthermore, the morphology changes were examined by atomic force microscopy (AFM), polarizable optical microscopy (POM), fluorescence microscopy, and dynamic light scattering (DLS) studies. These results provide very valuable clues on the Aß (CSF Aß fibrils, Aß1-40 fibrils, ß-amyloid lysozyme aggregates) disaggregation behavior via in vitro studies, which constitute the first insights into intracellular disaggregation of Aß by "turn-on" method thereby influencing amyloidogenesis.

Suicidal inactivation of methemoglobin by generation of thiyl radical: insight into NAC mediated protection in RBC.

N-acetyl-L-cysteine (NAC) improves antioxidant potentials of RBCs to provide protection against oxidative stress induced hemolysis. The antioxidant mechanism of NAC to reduce oxidative stress in RBC, studied through inactivation of pro-oxidant MetHb. NAC causes irreversible inactivation of the MetHb in an H2O2 dependent manner, and the inactivation follows the pseudo- first- order kinetics. The kinetic constants are ki = 8.5µM, kinact = 0.706 min(-1) and t1/2 = 0.9 min. Spectroscopic studies indicate that MetHb accepts NAC as a substrate and oxidizes through a single electron transfer mechanism to the NACox. The single e- oxidation product of NAC has been identified as the 5, 5'- dimethyl-1- pyrroline N- oxide (DMPO) adduct of the sulfur centered radical (a(N) = 15.2 G and a(H)=16.78 G). Binding studies indicate that NACox interacts at the heme moiety and NAC oxidation through MetHb is essential for NAC binding. Heme-NAC adduct dissociated from MetHb and identified (m/z 1011.19) as 2:1 ratio of NAC/heme in the adduct. TEMPO and PBN treatment reduces NAC binding to MetHb and protects against inactivation confirms the role of thiyl radical in the inactivation process. Furthermore, scavenging thiyl radicals by TEMPO abolish the protective effect of NAC in hemolysis. Current work highlights antioxidant mechanism of NAC through NAC thiyl radical generation, and MetHb inactivation to exhibit protection in RBC against oxidative stress induced hemolysis.

Methemoglobin incites primaquine toxicity through single-electron oxidation and modification.

BACKGROUND: Primaquine (Pq) metabolic products are responsible for drug-associated hemotoxicity and limit primaquine usage. METHODS: Methemoglobin (MetHb)-Pq molecular modeling was used to identify the Pq binding pocket. UPLC, mass spectrometry, and other indirect analytical methods were used to predict the metabolite. MetHb generation, development of oxidative stress, inhibition of antioxidant enzymes, and scanning electron microscope (SEM) were used to characterize the hemotoxic potentials of oxidized Pq (Pqox). RESULTS: MetHb binded Pq at the heme site with KD =6.4 µM as evidenced by a difference spectroscopy study. MetHb oxidized Pq through a single e-transfer mechanism to form Pqox. The analysis of Pq from MetHb-H2O2 peroxidase reaction mixture gave peaks at m/z 300.53 and m/z 243.42, corresponding to the hydroxyl and desamino derivative of Pq, respectively. Similar peaks were absent in Pq or Pq incubated with H2O2 in the same buffer system. A robust increase in MetHb formation, reactive oxygen species generation, and inhibition of antioxidant enzymes were found in red blood cells (RBCs) exposed to Pqox compared with a parent drug molecule. The RBC membrane exhibited visible damages to plasma membrane (holes) as evidenced by SEM analysis of Pqox-exposed RBCs. CONCLUSIONS: The MetHb-H2O2 system transforms quiescent parent drug molecule to a highly reactive oxidative form to exhibit severe hemolysis. MetHb-H2O2-mediated Pq hemolytic potentiation that is sensitive to spin trap indicates the role of Pq* radical or other single e-species in the process. The result suggests that MetHb incites the molecular property of the Pq and peroxidase inhibitors can be explored to control drug-associated toxicity.

Extracellular methemoglobin primes red blood cell aggregation in malaria: an in vitro mechanistic study.

Toxic byproducts from infected RBC cause rheological alteration and RBC aggregation. Malaria culture supernatant has the ability to exhibit RBC aggregation. Ammonium sulfate fractionation and immunodepletion of methemoglobin from culture supernatant confirms methemoglobin as a major aggregant. In vitro treatment of RBC with methemoglobin induces irreversible high order RBC aggregates, resistant to shear stress and physical forces. Methemoglobin-mediated ROS generation in the external micro-environment to develop oxidative stress close to RBC membrane seems to be responsible for initiating and forming high order RBC aggregates through phosphatidyl-serine externalization. Removal of oxidative stress through antioxidant treatment abolishes high order RBC aggregate formation. In conclusion, we discovered a novel pathway of methemoglobin-mediated RBC aggregation and its potential role in patho-physiological effects during malaria.

Extracellular Methemoglobin Mediated Early ROS Spike Triggers Osmotic Fragility and RBC Destruction: An Insight into the Enhanced Hemolysis During Malaria.

Malaria infection is known to cause severe hemolysis due to production of abnormal RBCs and enhanced RBC destruction through apoptosis. Infected RBC lysis exposes uninfected RBC to the large amount of pro-oxidant molecules such as methemoglobin. Methemoglobin (MetHb) exposure dose dependently makes RBCs susceptible to osmotic stress and causes hemolysis. MetHb mediated oxidative stress in RBC correlated well with osmotic fragility and hemolysis. Interestingly, a reactive oxygen species (ROS) spike at 15 min was responsible for the observed effects on RBC cells. Two natural antioxidants N-acetyl cysteine and mannitol protected the RBC from MetHb-mediated defects, which clearly indicated involvement of oxidative stress in the process. MetHb due to its pseudo-peroxidase activity produces ROS in the external microenvironment. Therefore, classical peroxidase inhibitors were tested to probe peroxidase activity mediated ROS production with defects in RBCs. Clotrimazole (CLT), which irreversibly inactivates the MetHb (CLT-MetHb) and abolishes peroxidase activity, did not produce significant ROS outside RBC and was inefficient to cause osmotic fragility and hemolysis. Hence, initiating a chain reaction, MetHb released from ruptured RBC produces significant ROS in the external microenvironment to make RBC membrane leaky and enhanced hemolysis. Together data presented in the current work explored the role of MetHb in accelerated humorless during malaria which could be responsible for severe outcomes of pathological disorders.