Hemoglobin Degradation Research Articles

The Blood Sugar Level Can Predict the Preoperative Shock in Patients With Ruptured Abdominal Aortic Aneurysm Even When the Patient's Condition Looks Stable

Introduction: This study aimed to identify the preoperative blood test data which can predict the preoperative shock in ruptured abdominal aortic aneurysm (rAAA). Methods: Total of 117 patients who underwent surgery including open repair and/or EVAR for rAAA between January 2007 and December 2018 were reviewed. The diagnosis of rAAA was made by computed tomography (CT) scan. Of these, 13 patients who underwent cardio pulmonary resuscitation at arrival, had been hospitalized for elective surgery for AAA, were complicated with chronic type B aortic dissection, and had been treated using the balloon occlusion at another hospital were excluded. In 104 patients (80% men, mean age 76 ± 10 years) with sufficient data, preoperative shock defined as a shock index (=heart rate/blood pressure) more than 1.5 or a maximum blood pressure less than 80 mmHg was observed in 43 patients (41%). Status of shock was categorized as follows; 1) “temporary shock before arrival” in 3 patients (7%) who had been treated by fluid resuscitation at community hospital and referred in stable condition, 2) “continuous shock” in 32 (74%) who arrived with shock that continued until aortic clamp, and 3) “shock after arrival” in 8 (19%) who arrived without shock but deteriorated into shock at emergency room in 6 and during CT scan in 2. To investigate the relation between shock and blood test data including blood sugar (BS), white blood cells, hemoglobin (Hb), platelets, C-reacting protein (CRP), creatinine, and fibrinogen degradation products in patients with rAAA, univariable and multivariable analyses were performed using the Logistic regression analysis. Results: The overall hospital mortality rate was 11% (11/104). BS (odds ratio [OR], 1.02; 95% confidence interval [CI]: 1.01-1.03; p=0.0001), CRP (OR, 0.56; 95% CI: 0.37-0.84; p=0.006), and Hb (OR, 0.63; 95% CI: 0.47-0.84; p=0.002) were identified as independent positive predictors of preoperative shock with multivariable analysis. The receiver operating characteristics curve (ROC) analysis for BS showed that area under the curve (AUC) for the predicted probabilities was 0.86 and at a cut-off value of 215 mg/dl, the sensitivity of the minimum BS level for predicting preoperative shock was 86% with a specificity of 79%. (Figure 1) The ROC curve for CRP showed the AUC of 0.74 and that for Hb presented the AUC of 0.69. BS level more than 215mg/dl were observed in 67% (2/3) patients with “temporary shock”, in 84% (27/32) with“continuous shock”, and in 100% (8/8) with“shock after arrival”. Conclusion: The BS level can be utilized as an independent predictor of preoperative shock in patients with rAAA. The patients having the preoperative BS level of 215mg/dl or more should be recognized as the patients to be treated emergently even when the patient's condition looks stable such as “temporary shock before arrival” or “shock after arrival”. Disclosure: Nothing to disclose

Toward a novel framework for bloodstains dating by Raman spectroscopy: How to avoid sample photodamage and subsampling errors

Answers to questions about the time of bloodstains formation are often essential to unravel the sequence of events behind criminal acts. Unfortunately, the relevance of preserved evidence to the committed offence usually cannot be verified, because forensic experts are still incapable of providing an accurate estimate of the bloodstains’ age. An antidote to this impediment might be substituting the classical dating approach – founded on the application of calibration models – by the comparison problem addressed using likelihood ratio tests. The key aspect of this concept involves comparing the evidential data with results characterizing reference bloodstains, formed during the process of supervised ageing so as to reproduce the evidence. Since this comparison requires data that conveys information inherent to changes accompanying the process of blood decomposition, this study provided a Raman-based procedure, designated for probing into the chemistry of ageing bloodstains. To circumvent limitations experienced with single-point measurements – the risk of laser-induced degradation of hemoglobin and subsampling errors – the rotating mode of spectral acquisition was introduced. In order to verify the performance of this novel sampling method, obtained spectra were confronted with those acquired during conventional static measurements. The visual comparison was followed by analysis of data structure using regularized MANOVA, which boosted the variance between differently-aged samples while minimizing the variance observed for bloodstains deposited at the same time. Studies of relation between these variances demonstrated the superiority of novel procedure, as it provided Raman signatures that enabled a better distinction between differently-aged bloodstains.

Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi

Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomine's midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while in trypomastigotes an increase in intracellular cAMP doesn't seem to aid in this response, although it does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

South African Abietane Diterpenoids and Their Analogs as Potential Antimalarials: Novel Insights from Hybrid Computational Approaches.

The hemoglobin degradation process in Plasmodium parasites is vital for nutrient acquisition required for their growth and proliferation. In P. falciparum, falcipains (FP-2 and FP-3) are the major hemoglobinases, and remain attractive antimalarial drug targets. Other Plasmodium species also possess highly homologous proteins to FP-2 and FP-3. Although several inhibitors have been designed against these proteins, none has been commercialized due to associated toxicity on human cathepsins (Cat-K, Cat-L and Cat-S). Despite the two enzyme groups sharing a common structural fold and catalytic mechanism, distinct active site variations have been identified, and can be exploited for drug development. Here, we utilize in silico approaches to screen 628 compounds from the South African natural sources to identify potential hits that can selectively inhibit the plasmodial proteases. Using docking studies, seven abietane diterpenoids, binding strongly to the plasmodial proteases, and three additional analogs from PubChem were identified. Important residues involved in ligand stabilization were identified for all potential hits through binding pose analysis and their energetic contribution determined by binding free energy calculations. The identified compounds present important scaffolds that could be further developed as plasmodial protease inhibitors. Previous laboratory assays showed the effect of the seven diterpenoids as antimalarials. Here, for the first time, we demonstrate that their possible mechanism of action could be by interacting with falcipains and their plasmodial homologs. Dynamic residue network (DRN) analysis on the plasmodial proteases identified functionally important residues, including a region with high betweenness centrality, which had previously been proposed as a potential allosteric site in FP-2.

A comprehensive review of bilirubin determination methods with special emphasis on biosensors

Abstract Bilirubin, is a tetrapyrrole yellow coloured compound found in digestive juice. It is generated from degradation of hemoglobin (Hb). The normal range of total bilirubin in serum is 0.30–1.20 mg/dl. The elevated range of serum bilirubin is considered as biomarker for finding and therapeutic administration of many liver diseases. Various analytical methods for determination of bilirubin, including spectrophotometery, thin layer chromatography, fluorometry, capillary electrophoresis, high performance liquid chromatographic, polarography and chemiluminescence have been applied for clinical purposes. These conventional methods are tedious, time-consuming, and require costly equipments and skilled person to operate. To overcome these limitations, the most popular biosensing technology has been employed at a large scale. The present review describes the principle, advantages and disadvantages of different analytic methods for measurement of bilirubin with focusing on biosensors, including electrochemical, photo-electrochemical, piezoelectric, optical and luminescent biosensors in detail. The working conditions for optimum activity and shelf life of all bilirubin biosensors have been summarized & compared and their future perspectives are discussed.

Current Challenges in the Development of Acellular Hemoglobin Oxygen Carriers by Protein Engineering.

This article reviews the key biochemical mechanisms that govern O2 transport, NO scavenging, and oxidative degradation of acellular hemoglobin (Hb) and how these ideas have been used to try to develop strategies to engineer safer and more effective hemoglobin-based oxygen carriers (HBOCs). Significant toxicities due to acellular Hb have been observed after the administration of HBOCs or after the lysis of red cells, and include rapid clearance and kidney damage due to dissociation into dimers, haptoglobin binding, and macrophage activation; early O2 release leading to decreased tissue perfusion in capillary beds; interference with endothelial and smooth muscle signaling due to nitric oxide (NO) scavenging; autooxidization of heme iron followed by production of reactive oxygen species; and iron overload symptoms due to hemin loss, globin denaturation, iron accumulation, and further inflammation. Protein engineering can be used to mitigate some of these side effects, but requires an in-depth mechanistic understanding of the biochemical and biophysical features of Hb that regulate quaternary structure, O2 affinity, NO dioxygenation, and resistance to oxidation, hemin loss, and unfolding.

The effects of nickel oxide nanoparticles on structural changes, heme degradation, aggregation of hemoglobin and expression of apoptotic genes in lymphocytes

Nickel oxide nanoparticles (NiO NPs) have received great interests in medical and biotechnological applications. However, their adverse impacts against biological systems have not been well-explored. Herein, the influence of NiO NPs on structural changes, heme degradation and aggregation of hemoglobin (Hb) was evaluated by UV-visible (Vis) spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), and molecular modeling investigations. Also, the morphological changes and expression of Bax/Bcl-2 mRNA in human lymphocyte cell exposed to NiO NPs were assayed by DAPI staining and quantitative real-time PCR (qPCR), respectively. The UV-Vis study depicted that NiO NPs resulted in the displacement of aromatic residues and heme groups and production of the pro-aggregatory species. Intrinsic and Thioflavin T (ThT) fluorescence studies revealed that NiO NPs resulted in heme degradation and amorphous aggregation of Hb, respectively, which the latter result was also confirmed by TEM study. Moreover, far UV-CD study depicted that NiO NPs lead to substantial secondary structural changes of Hb. Furthermore, near UV-CD displayed that NiO NPs cause quaternary conformational changes of Hb as well as heme displacement. Molecular modelling study also approved that NiO NPs resulted in structural alterations of Hb and heme deformation. Moreover, morphological and genotoxicity assays revealed that the DNA fragmentation and expression ratio of Bax/Bcl-2 mRNA increased in lymphocyte cells treated with NiO NPs for 24 hr. In conclusion, this study indicates that NiO NPs may affect the biological media and their applications should be limited.Communicated by Ramaswamy H. Sarma

Evaluation of the usefulness of the simultaneous assay of fecal hemoglobin (Hb) and transferrin (Tf) in colorectal cancer screening – for the establishment of the Hb and Tf two-step cutoff assay (HTTC assay)

Background One of the issues of fecal immunochemical test (FIT) is false-negativity caused by hemoglobin degradation by bacteria. We investigated the usefulness of the transferrin assay, which is stable toward bacteria. Methods The study included 1174 patients who visited our hospital and underwent colonoscopy for some symptoms or for cancer screening. We compared the hemoglobin-transferrin combination assay with the hemoglobin-alone assay using the Discrete Clinical Chemistry Analyzer NS-Plus and 1174 clinical samples. In the combination assay, two hemoglobin cutoff values (a) and (b) and one transferrin cutoff value (c) were set. Cases with values of (a) or more were defined as primary positive and values lower than (b) were defined as negative. Cases with values between (a) and (b) underwent the transferrin assay. Then, cases with values of (c) or higher were defined as secondary positive. All primary and secondary positive cases were defined as positive. Among the combination of cutoff values (a), (b) and (c), we identified Method A exhibiting high specificity and a positive predictive value (PPV), and Method B exhibiting the highest sensitivity. Results In Method A, the sensitivity of colorectal cancer detection increased from 67.3% to 68.2%, the specificity significantly (p = 0.0011) increased from 90.5% to 92.6%, and the PPV increased from 42.9% to 49.6% compared with the hemoglobin-alone assay. In Method B, the sensitivity increased significantly (p = 0.046) from 67.3% to 71.1% and the PPV increased from 42.9% to 44.8%. Conclusions This combination assay showed higher accuracy and effectiveness for colorectal cancer screening.

Melatonin Prevents Mice Cortical Astrocytes From Hemin-Induced Toxicity Through Activating PKCα/Nrf2/HO-1 Signaling in vitro.

Secondary injuries mediated by oxidative stress lead to deterioration of neurological functions after intracerebral hemorrhage (ICH). Cortical astrocytes are among the most important cells in the central nervous system (CNS), and play key roles in maintaining redox homeostasis by providing oxidative stress defense. Hemin is a product of hemoglobin degradation, which has strong toxicity and can induce reactive oxygen species (ROS). Melatonin (Mel) and its metabolites are well tolerated without toxicity, prevent tissue damage as well as effectively assist in scavenging free radicals. We evaluated the hemin neurotoxicity to astrocytes and the resistance of Mel-treated astrocytes to hemin neurotoxicity. And we found Mel induced PKCα phosphorylation (p-PKC), nuclear translocation of Nrf2 in astrocytes, and upregulation of HO-1, which contributed to the reduction of ROS accumulation and cell apoptosis. Nrf2 and HO1 protein expression upregulated by Mel were decreased after administration of PKC inhibitor, Ro 31-8220 (Ro 31). Luzindole (Luz), a melatonin receptor inhibitor, suppressed p-PKCα, HO-1, and Nrf2 expression upregulated by Mel and increased cell apoptosis rate. The upregulation of HO-1 induced by Mel was depressed by knocking down Nrf2 expression by siRNA, which also decreased the resistance of astrocytes to toxicity of hemin. Mel activates astrocytes through PKCα/Nrf2/HO-1 signaling pathway to acquire resistance to toxicity of hemin and resist from oxidative stress and apoptosis. The positive effect of Mel on PKCα/Nrf2/HO-1 signaling pathway may become a new target for neuroprotection after intracerebral hemorrhage.

Oxidative Stress and Hemoglobin Level of Complicated and Uncomplicated Malaria Cases among Children: A Cross-Sectional Study in Kumasi Metropolis, Ghana.

Introduction Malaria is a leading cause of mortality among children below 5 years in Ghana. Its parasites are known to cause the degradation of hemoglobin, resulting in the production of reactive oxygen species and hence oxidant stress. Therefore, this study was carried out to compare the levels of oxidative stress between children with complicated and uncomplicated malaria infection in Kumasi, Ghana. Method Subjects were recruited from hospitals in the Kumasi Metropolis. This was a cross-sectional study, involving 17 complicated malaria subjects, 51 uncomplicated malaria subjects, and 15 nonparasitemic subjects. The rapid diagnostic test (RDT) was used to determine presence or absence of falciparum malaria among the study participants. Blood samples from subjects were used to determine hemoglobin, malondialdehyde (MDA), and vitamin C levels. Results Majority of the subjects (67.5%) were within the age of 0-5 years. The mean age (±SD) of uncomplicated malaria subjects was 4.32 (±2.81) years, while that of complicated malaria was 4.27 (±2.96). Mean levels of HB decreased significantly in the following order: control subjects > uncomplicated malaria subjects > complicated malaria subjects (p<0.0001). Mean levels of MDA were significantly lower in control subjects compared to complicated malaria subjects (4.62±1.85 versus 6.68±0.70, p=0.0008) and also lowered in uncomplicated malaria subjects compared to complicated malaria (4.50±1.58 versus 6.68±0.70, p<0.0001). There was a statistically significant reduced mean level of vitamin C (p=0.036) in the following order: control subjects > uncomplicated malaria > complicated malaria subjects. However, for the complicated malaria cases, there were significantly higher mean vitamin C levels in females than in males (p<0.001). Conclusion Malaria progression increases MDA levels and decreases the ascorbate (vitamin C) and hemoglobin levels. It is recommended that future studies should investigate changes in other antioxidant vitamins, like vitamins A and E.

Optical tweezers combined with micro‐Raman investigation of alcohol‐induced changes on single, live red blood cells in blood plasma

AbstractAlcohol is commonly used as sterilizing agent in hospital settings. Alcohol abuse is also associated with numerous medical complications. It influences the coagulation mechanics and erythropoiesis. Optical tweezers are being extensively used in research labs for the trapping as well as manipulation of biological cells. The biochemical investigation of trapped biological cells can be realized with the integration of Raman spectroscopic technique with optical tweezers, which is termed as Raman tweezers. In this work, we interrogate the influence of ethanol exposure on human red blood cells at single cell level using micro‐Raman spectroscopy. In order to mimic in vivo condition to an extent, the experiment is performed by optically trapping the live cell in blood plasma. In the present work, a 785‐nm diode laser was used for trapping and probing of single, live red blood cell. An intensity deduction was noted for porphyrin breathing mode at 752 cm‐1 in the Raman spectra as a function of ethanol concentration, which indicates the depletion in hemoglobin. Hemoglobin deoxygenation process in the presence of alcohol is also evident from the variations in major oxygenation marker bands at 1,209; 1,222; 1,544; 1,561 cm‐1, etc., in the Raman spectrum of red blood cell. The effect of alcohol on red blood cells in blood plasma induces membrane depletion and hemoglobin degradation. This is also accompanied by the conversion of oxygenated hemoglobin to more deoxygenated state. Present study has also demonstrated the capability of micro‐Raman spectroscopy as a promising tool for probing red blood cell oxygenation status.

Hemin-induced platelet activation and ferroptosis is mediated through ROS-driven proteasomal activity and inflammasome activation: Protection by Melatonin

Reactive oxygen species (ROS) are capable of inducing cell death or apoptosis. Recently, we demonstrated that lipid-ROS can mediate ferroptosis and activation of human platelets. Ferroptosis is an intracellular iron-mediated cell death, distinct from classical apoptosis and necrosis, which is mediated through the accumulation of ROS, lipid peroxides and depletion of cellular GSH. Lately, we demonstrated that hemoglobin degradation product hemin induces ferroptosis in platelets via ROS and lipid peroxidation. In this study, we demonstrate that hemin-induced ferroptosis in platelets is mediated through ROS-driven proteasome activity and inflammasome activation, which were mitigated by Melatonin (MLT). Although inflammasome activation is linked with pyroptosis, it is still not clear whether ferroptosis is associated with inflammasome activation. Our study for the first time demonstrates an association of platelet activation/ferroptosis with proteasome activity and inflammasome activation. Although, high-throughput screening has recognized ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as potent ferroptosis inhibitors, having an endogenous antioxidant such as MLT as ferroptosis inhibitor is of high interest. MLT is a well-known chronobiotic hormone that regulates the circadian rhythms in vertebrates. It also exhibits potent antioxidant and ROS quenching capabilities. MLT can regulate fundamental cellular functions by exhibiting cytoprotective, oncostatic, antiaging, anti-venom, and immunomodulatory activities. The ROS scavenging capacity of MLT is key for its cytoprotective and anti-apoptotic properties. Considering the anti-ferroptotic and anti-apoptotic potentials of MLT, it could be a promising clinical application to treat hemolytic, thrombotic and thrombocytopenic conditions. Therefore, we propose MLT as a pharmacological and therapeutic agent to inhibit ferroptosis and platelet activation.

Blood anticlotting activity of a Rhipicephalus microplus cathepsin L-like enzyme

In parasites, cathepsins are implicated in mechanisms related to organism surveillance and host evasion. Some parasite cathepsins have fibrinogenolytic and fibrinolytic activity, suggesting that they may contribute to maintain blood meal fluidity for extended feeding periods. Here, it is shown that BmGTI (Rhipicephalus [Boophilus] microplus Gut Thrombin Inhibitor), a protein previously described as an inhibitor of fibrinogen hydrolysis and platelet aggregation by thrombin, and BmCL1 (Rhipicephalus [Boophilus] microplus Cathepsin-L like 1) are the same protein, hereinafter referred to using the earliest name (BmCL1). To further characterize BmCL1, Rhipicephalus microplus native and recombinant (rBmCL1) proteins were obtained. Native BmCL1 was isolated using thrombin-affinity chromatography, and it displays thrombin inhibition activity. We subsequently investigated rBmCL1 interaction with thrombin. We show that rBmCL1 and thrombin have a dissociation constant (ΚD) of 130.2 ± 11.2 nM, and this interaction likely occurs due to a more electronegative surface of BmCL1 at pH 7.5 than at pH 5.0, which may favor an electrostatic binding to positively charged thrombin exosites. During BmCL1-thrombin interaction, thrombin is not degraded or inhibited. rBmCL1 impairs thrombin-induced fibrinogen clotting via a fibrinogenolytic activity. Fibrinogen degradation by BmCL1 occurs by the hydrolysis of Aα- and Bβ-chains, generating products similar to those produced by fibrinogenolytic cathepsins from other organisms. In conclusion, BmCL1 likely has an additional role in R. microplus blood digestion, besides its role in hemoglobin degradation at acid pH. BmCL1 fibrinogenolytic activity indicates a proteolytic activity in the neutral lumen of tick midgut, contributing to maintain the fluidity of the ingested blood, which remains to be confirmed in vivo.

Comparing sequence and structure of falcipains and human homologs at prodomain and catalytic active site for malarial peptide based inhibitor design

BackgroundFalcipains are major cysteine proteases of Plasmodium falciparum involved in haemoglobin degradation and remain attractive anti-malarial drug targets. Several inhibitors against these proteases have been identified, yet none of them has been approved for malaria treatment. Other Plasmodium species also possess highly homologous proteins to falcipains. For selective therapeutic targeting, identification of sequence and structure differences with homologous human cathepsins is necessary. The substrate processing activity of these proteins is tightly controlled via a prodomain segment occluding the active site which is chopped under low pH conditions exposing the catalytic site. Current work characterizes these proteases to identify residues mediating the prodomain regulatory function for the design of peptide based anti-malarial inhibitors.MethodsSequence and structure variations between prodomain regions of plasmodial proteins and human cathepsins were determined using in silico approaches. Additionally, evolutionary clustering of these proteins was evaluated using phylogenetic analysis. High quality partial zymogen protein structures were modelled using homology modelling and residue interaction analysis performed between the prodomain segment and mature domain to identify key interacting residues between these two domains. The resulting information was used to determine short peptide sequences which could mimic the inherent regulatory function of the prodomain regions. Through flexible docking, the binding affinity of proposed peptides on the proteins studied was evaluated.ResultsSequence, evolutionary and motif analyses showed important differences between plasmodial and human proteins. Residue interaction analysis identified important residues crucial for maintaining prodomain integrity across the different proteins as well as the pro-segment responsible for inhibitory mechanism. Binding affinity of suggested peptides was highly dependent on their residue composition and length.ConclusionsDespite the conserved structural and catalytic mechanism between human cathepsins and plasmodial proteases, current work revealed significant differences between the two protein groups which may provide valuable information for selective anti-malarial inhibitor development. Part of this study aimed to design peptide inhibitors based on endogenous inhibitory portions of protease prodomains as a novel aspect. Even though peptide inhibitors may not be practical solutions to malaria at this stage, the approach followed and results offer a promising means to find new malarial inhibitors.

New Method of Production and Characterization of Haemozoin and Β-Haemozoin from Meccus longipennis

Understanding the significance of hemozoin (Hz) in the process through which Plasmodium is released from the heme group in the food vacuole during hemoglobin degradation, will allow the development of more effective drugs against malaria. Therefore, the development of methodologies to obtain Hz synthetically will facilitate an in vitro evaluation of new anti-malarial drugs. We present a methodology with good results to obtain Hz from fecal material of blood-sucking insects Meccus longipennis. The preparation of biological cultures of the parasite (Plasmodium) transmitter of the disease is not necessary. The hemozoin molecule and its dimer were obtained using the method described and it was possible to validate a comparison with the positive and negative controls using different analytical techniques. The proposed method allows obtaining hemozoin and its dimer demonstrating equivalence with positive controls that demonstrate that the present procedure may be an alternative for the evaluation of antimalarial drugs.

In silico study of M18 aspartyl amino peptidase (M18AAP) of Plasmodium vivax as an antimalarial drug target.

Plasmodium vivax (Pv) is the second most malaria causing pathogen among Plasmodium species. M18 aspartic aminopeptidase (M18AAP) protein is a single gene copy present in Plasmodium. This protein is functional at the terminal stage of hemoglobin degradation of host and completes the hydrolysis process which makes it an important target for new chemotherapeutics. No experimental and structural study on M18AAP protein of P. vivax is reported till today. This paper advocates the application of multiple computational approaches like protein model prediction, ligand-based 3D QSAR study, pharmacophore, structure-based virtual screening and molecular docking simulation for identification of potent lead molecules against the enzyme. The 3D QSAR model was developed using known bioactive compounds against the PvM18AAP protein which statistically signify the k-NN model with q^2 = 0.7654. The study reports a lead molecule from ligand-centric approach with good binding affinity and possessing lowest docking score. The findings will be helpful for in-vivo and in-vitro validations and development of potent anti-malarial molecules against the drug resistant strains of malaria parasite.

Behavioral effects evoked by the beta globin-derived nonapeptide LVV-H6

LVV-hemorphin-6 (LVV-h6) is bioactive peptide and is a product of the degradation of hemoglobin. Since LVV-h6 effects are possibly mediated by opioid or AT4/IRAP receptors, we hypothesized that LVV-h6 would modify behavior. We evaluated whether LVV-h6 affects: i) anxiety-like behavior and locomotion; ii) depression-like behavior; iii) cardiovascular and neuroendocrine reactivity to emotional stress. Male Wistar rats ( ± 300 g) received LVV-h6 (153 nmol/kg i.p.) or vehicle (NaCl 0.9% i.p.). We used: i) open field (OF) test for locomotion; ii) elevated plus maze (EPM) for anxiety-like behavior; iii) forced swimming test (FST) for depression-like behavior and iv) air jet for cardiovascular and neuroendocrine reactivity to stress. Diazepam (2 mg/kg i.p.) and imipramine (15 mg/kg i.p.) were used as positive control for EPM and FST, respectively. To evaluate the LVV-h6 mechanisms, we used: the antagonist of oxytocin (OT) receptors (atosiban – ATS 1 and 0.1 mg/kg i.p.); the inhibitor of tyrosine hydroxylase (Alpha-methyl-p-tyrosine – AMPT 200 mg/kg i.p.) to investigate the involvement of catecholaminergic paths; and the antagonist of opioid receptors (naltrexone – NTX 0.3 mg/kg s.c.). We found that LVV-h6: i) evoked anxiolytic-like effect; ii) evoked antidepressant-like effect in the FST; and iii) did not change the locomotion, neuroendocrine and cardiovascular responses to stress. The LVV-h6 anxiolytic-like effect was not reverted by ATS and AMPT. However, the antidepressant effects were reverted only by NTX. Hence, our findings demonstrate that LVV-h6 modulates anxiety-like behavior by routes that are not oxytocinergic, catecholaminergic or opioid. The antidepressant-like effects of LVV-h6 rely on opioid pathways.

Multimodal analysis of Plasmodium knowlesi-infected erythrocytes reveals large invaginations, swelling of the host cell, and rheological defects.

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block‐face scanning electron microscopy to explore the topography of P. knowlesi‐infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi‐infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python‐based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi‐infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.

Non-immunological toxicological mechanisms of metamizole-associated neutropenia in HL60 cells

Metamizole is an analgesic and antipyretic, but can cause neutropenia and agranulocytosis. We investigated the toxicity of the metabolites N-methyl-4-aminoantipyrine (MAA), 4-aminoantipyrine (AA), N-formyl-4-aminoantipyrine (FAA) and N-acetyl-4-aminoantipyrine (AAA) on neutrophil granulocytes and on HL60 cells (granulocyte precursor cell line). MAA, FAA, AA, and AAA (up to 100 µM) alone were not toxic for HL60 cells or granulocytes. In the presence of the myeloperoxidase substrate H2O2, MAA reduced cytotoxicity for HL60 cells at low concentrations (<50 µM), but increased cytotoxicity at 100 µM H2O2. Neutrophil granulocytes were resistant to H2O2 and MAA. Fe2+ and Fe3+ were not toxic to HL60 cells, irrespective of the presence of H2O2 and MAA. Similarly, MAA did not increase the toxicity of lactoferrin, hemoglobin or methemoglobin for HL60 cells. Hemin (hemoglobin degradation product containing a porphyrin ring and Fe3+) was toxic on HL60 cells and cytotoxicity was increased by MAA. EDTA, N-acetylcystein and glutathione prevented the toxicity of hemin and hemin/MAA. The absorption spectrum of hemin changed concentration-dependently after addition of MAA, suggesting an interaction between Fe3+ and MAA. NMR revealed the formation of a stable MAA reaction product with a reaction pathway involving the formation of an electrophilic intermediate. In conclusion, MAA, the principle metabolite of metamizole, increased cytotoxicity of hemin by a reaction involving the formation of an electrophilic metabolite. Accordingly, cytotoxicity of MAA/hemin could be prevented by the iron chelator EDTA and by the electron donors NAC and glutathione. Situations with increased production of hemin may represent a risk factor for metamizole-associated granulocytopenia.