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After a century of constant failure to produce an in vitro culture of the most widespread human malaria parasite Plasmodium vivax, recent advances have highlighted the difficulties to provide this parasite with a healthy host cell to invade, develop, and multiply under in vitro conditions. The actual level of understanding of the heterogeneous populations of cells—framed under the name ‘reticulocytes’—and, importantly, their adequate in vitro progression from very immature reticulocytes to normocytes (mature erythrocytes) is far from complete. The volatility of its individual stability may suggest the reticulocyte as a delusory cell, particularly to be used for stable culture purposes. Yet, the recent relevance gained by a specific subset of highly immature reticulocytes has brought some hope. Very immature reticulocytes are characterized by a peculiar membrane harboring a plethora of molecules potentially involved in P. vivax invasion and by an intracellular complexity dynamically changing upon its quick maturation into normocytes. We analyze the potentialities offered by this youngest reticulocyte subsets as an ideal in vitro host cell for P. vivax.

Incomplete non-sterile immunity to malaria is attained in endemic regions after recurrent infections by a large percentage of the adult population, who carry the malaria parasite asymptomatically. Although blood-stagePlasmodium falciparumrapidly elicits IgG responses, the target antigens of partially protective and non-protective IgG antibodies as well as the basis for the acquisition of these antibodies remain largely unknown. We performed IgG-immunomics to screen forP. falciparumantigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients’ IgGs. For further epitope mapping, peptide microarrays designed to cover their sequences were probed with a set of 38 sera samples from adult individuals of an endemic malaria region in Ghana. Eight 20-mer peptides with the highest affinity and frequency of recognition among the population were subsequently validated with 16 sera from the same region, segregated into patients with positive or negative subclinical detection ofP. falciparum. Significant binding specificity for two immunodominant antigenic regions was uncovered within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer peptides challenged with sera samples from children under 5 years old displayed specific IgG binding in those with detectable parasitemia, even at subclinical level. These results suggest that the humoral response against START and PDI8 antigens may be triggered even at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.SignificanceMalaria in Africa is a leading cause of morbidity and mortality. The reservoirs of the malaria parasite are asymptomatic patients who carry it subclinically. Identifying the parasite antigens and its fragments that trigger the most common immunity response by immunoglobulin G that partially protect people can have profound implications for both, development of a malaria vaccine and diagnosis of the subclinical parasite carriers. Antigen discovery and mapping, validated with sera from subclinical carriers, showed that immunoglobulin G responses in children against parasite’s START and PDI8 may eventually be used to differentially diagnose non-infected from subclinical cases. Furthermore, anti-START and anti-PDI8 endemic immunodominance provides association of these antigens with long-term acquired immunity and immune evasion to malaria.

Within the pathophysiology of epilepsy, as a chronic brain disorder, the involvement of neuroinflammation has been extensively implied. Recurrent seizures of epilepsy have been associated with elevated levels of immune mediators that seem to play a pivotal role in triggering them. Neurons, glia, and endothelial cells of the blood-brain barrier (BBB) take part in such inflammatory processes by expressing receptors of associated mediators through autocrine and paracrine stimulation of intracellular signaling pathways. In this milieu, elevated cytokine levels in serum and brain tissue have been reported in patients with an epileptic profile. Noteworthy, interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) are the proinflammatory cytokines mostly associated, in literature, with the pathogenesis of epilepsies. In this review, we examine the function of these cytokines in connection with transforming growth factor-beta (TGF-β), IL-8, IL-12, IL-18, and macrophage inflammatory protein (MIP) as potential proinflammatory mediators in the neuropathology of epilepsy.

The T helper 17 (Th17) cells contain a dynamic subset of CD4+ T-cells that are able to develop into other different lineage subsets, including the Th1-like Th17 cells. These cells co-express retinoic acid-related orphan receptor gamma t (RORγt) and transcription factor T-box-expressed-in-T-cells (T-bet) and produce both interleukin (IL)-17 and interferon (IFN)-γ. Recent reports have shown that Th1-like Th17 cells play crucial roles in the pathogenesis of autoimmune diseases such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, as well as, some primary immunodeficiency with autoimmune features. Here, the actual mechanisms for Th17 cells plasticity to Th1-like Th17 cells are discussed and reviewed in association to the role that Th1-like Th17 cells have on inflammatory and autoimmune disorders.

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M−1s−1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.

The neurotrophin b-nerve growth factor (NGF) has been described as an important mediator of seminal plasma-induced ovulation in reflexive ovulators like camelids, but this action has not been evidenced in rabbits. In this study, the mRNA expression of NGF was analysed in rabbit male organs by PCR and in a purified basic fraction of seminal plasma by liquid chromatography mass spectrometry. Besides, the ovulatory response [ovulation rate (OR), corpora lutea number, LH peak and progesterone profile] of 27 rabbit does was recorded after injection with murine NGF, gonadoreline (GnRH) or saline solution (SS). An empty catheter was introduced through the vagina (n ¼ 4 animals per group) to simulate the nervous/mechanical stimulus of coitus. Molecular studies confirmed the expression of b-NGF mRNA in the prostate, seminal gland and testicles of male adult rabbits, and the presence of the protein in seminal plasma. For the in vivo experiment, all GnRH-group does exhibited a significantly higher OR (100%) compared to females treated with NGF (30%) and SS (25%), who showed significantly lower LH and progesterone concentrations at 120 min. However, in the non-vaginally stimulated animals, only the gonadoreline group ovulated properly, since no ovulation was recorded in the SS group, and only 17% of the NGF does ovulated; this was associated to a tendency to a higher rate of anovulatory haemorrhagic follicles. mRNA expression of NGF and its presence in seminal plasma evidence a role in rabbit reproduction. Nevertheless, murine NGF triggered some stimulatory effect in the females but not enough to elicit a significant ovulatory response.

Control of oxidative stress in the bone marrow (BM) is key for maintaining the interplay between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an indicator of oxidative stress in the BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox (DFX). As expected, differences in the pattern of protein carbonylation were observed in BM samples between MDS patients and controls, with an increase in protein carbonylation in the former. Strikingly, patients under DFX treatment had lower levels of protein carbonylation in BM with respect to untreated patients. Proteomic analysis identified four proteins with high carbonylation levels in MDS BM cells. Finally, as oxidative stress-related signaling pathways can modulate the cell cycle through p53, we analyzed the expression of the p53 target gene p21 in BM cells, finding that it was significantly upregulated in patients with MDS and was significantly downregulated after DFX treatment. Overall, our results suggest that the fine-tuning of oxidative stress levels in the BM of patients with MDS might control malignant progression.

Background:Recent advances in antiviral therapy show potential for a cure and/or control of most human infections caused by hepatitis viruses and retroviruses. However, medical success is largely dependent on the identification of the large number of people unaware of these infections, especially in developing countries. Dried blood spots (DBS) have been demonstrated to be a good tool for collecting, storing and transporting clinical specimens from rural areas and limited-resource settings to laboratory facilities, where viral infections can be more reliably diagnosed.Methods:The seroprevalence and virological characterization of hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as human retroviruses (HIV-1, HIV-2, human T-cell leukaemia virus type 1 [HTLV-1] and human T-cell leukaemia virus type 2 [HTLV-2]), were investigated in clinical specimens collected from DBS in Ghana.Results:A total of 305 consecutive DBS were collected. A high prevalence of chronic HBV (8.5%) and occult hepatitis B (14.2%) was found, whereas rates were lower for HIV-1, HTLV-1 and HCV (3.2%, 1.3% and 0.6%, respectively). HIV-2 and HTLV-2 were absent. CRF02_AG was the predominant HIV-1 subtype, whereas genotype E was the most frequent HBV variant.Conclusions:DBS are helpful in the diagnosis and virological characterization of hepatitis and retrovirus infections in resource-limited settings. The high rate of hepatitis B in Ghana, either overt or occult, is noteworthy and confirms recent findings from other sub-Saharan countries. This should encourage close clinical follow up and antiviral treatment assessment in this population, as well as universal HBV vaccine campaigns.

Malaria is a parasitic disease caused by Plasmodium spp., being one of the major causes of death worldwide with two-hundred million new infections and hundreds of thousands of deaths in 2015. Despite the important advances in its prevention and treatment, its resistance to current drug therapies is still a serious risk in its eradication. There is urgency in finding novel targets and drugs operating by novel mechanisms, avoiding cross-resistance to classical antimalarials. In this context, the bifunctional enzyme Glucose-6- phosphate dehydrogenase 6-phosphogluconolactonase appears to be a promising therapeutic target due to its crucial role in regulating the PPP pathway (pentose phosphate pathway), which is the major source of redox potential in Plasmodium falciparum. In the last few years, our group detected a specific mutation between the human and the Plasmodium falciparum form in the binding site of Glucose-6-phosphate (G6P), the endogenous ligand of Glucose-6-phosphate dehydrogenase (G6PD). This mutation involves the substitution of an Arginine (human) by an Aspartate (parasite), which allowed us to create a validated in-house homology model of PfG6PD. Based on this result, the group has focused their efforts, through different molecular modelling techniques, in the discovery of selective scaffolds against PfG6PD. Current efforts address the development of a complete structural model of the bifunctional enzyme, which may offer novel opportunities to develop molecules capable of inhibiting this relevant enzyme.

In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.

This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits.

β-Nerve Growth Factor (β-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of β-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. β-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine β-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate β-NGF concentration, and testosterone levels were also assessed in BP. The highest β-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual β-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of β-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, β-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. β-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and β-NGF.

Quinones are a family of bioactive compounds with known antibacterial, antiviral and antiparasitic activity. The capacity of quinoid compounds to accept electrons, due to electron-attracting (or donating) substituents at the quinone moiety, modulates their redox activity upon interaction with biological systems. Nine quinone derivate including three 2 - hydroxyl - 1, 4 - naphthoquinone; three 4,5 - and three 4,9 - naphthoquinone scaffolds, were synthesized and evaluated against 3D7 and Dd2 Plasmodium falciparum strains and evaluated the oxidative stress. Cell viability was determined MTT-related colorimetric assay (EZ4u; Biomedical, Austria). Selectivity of these compounds was evaluated by comparing in vitro, of cytotoxicity in human fibroblast and antimalarial activity in Plasmodium falciparum. Peripheral blood mononuclear cells and hemoglobin release from human erythrocytes were evaluated too. Antiplasmodial activity was evaluated by fluorometry methods. In addition, oxidized membrane protein in the malaria parasite was evaluated in vitro by derivatization, after treatment at IC50 values on P. falciparum Dd2 strain. Quinones showed IC50 values in the micromole range, selectivity indexes for quinones was variable, compounds 2 and 3 showed excellent selectivity. In vitro antiplasmodial activity was showed; this result is an incentive to continue synthesis and studying the quinoid compounds. Key words: Malaria, Quinones, Plasmodium falciparum.

The co-endemicity of malnutrition, erythrocytopathies, transmissible diseases and iron-deficiency contribute to the prevalence of chronic anaemia in many populations of the developing world. Although iron dietary supplementation is applied or recommended in at risk populations, its use is controversial due to undesirable outcomes, particularly regarding the response to infections, including highly prevalent malaria. We hypothesized that a boosted oxidative stress due to iron supplementation have a similar impact on malaria to that of hereditary anaemias, enhancing innate response and conditioning tissues to prevent damage during infection. Thus, we have analysed antioxidant and innate responses against lethal Plasmodium yoelii during the first five days of infection in an iron-supplemented mouse. This murine model showed high iron concentration in plasma with upregulated expression of hemoxygenase-1. The sustained homeostasis after this extrinsic iron conditioning, delayed parasitemia growth that, once installed, developed without anaemia. This protection was not conferred by the intrinsic iron overload of hereditary hemochromatosis. Upon iron-supplementation, a large increase of the macrophages/dendritic cells ratio and the antigen presenting cells was observed in the mouse spleen, independently of malaria infection. Complementary, malaria promoted the splenic B and T CD4 cells activation. Our results show that the iron supplementation in mice prepares host tissues for oxidative-stress and induces unspecific cellular immune responses, which could be seen as an advantage to promote early defences against malaria infection.

Proteomics is improving malaria research by providing global information on relevant protein sets from the parasite and the host in connection with its cellular structures and specific functions. In the last decade, reports have described biologically significant elements in the proteome of Plasmodium, which are selectively targeted and quantified, allowing for sensitive and high-throughput comparisons. The identification of molecules by which the parasite and the host react during the malaria infection is crucial to the understanding of the underlying pathogenic mechanisms. Hence, proteomics is playing a major role by defining the elements within the pathogenic space between both organisms that change across the parasite life cycle in association with the host transformation and response. Proteomics has identified post-translational modifications in the parasite and the host that are discussed in terms of functional interactions in malaria parasitism. Furthermore, the contribution of proteomics to the investigation of immunogens for potential vaccine candidates is summarized. The malaria-specific technological advances in proteomics are particularly suited now for identifying host-parasite interactions that could lead to promising targets for therapy, diagnosis or prevention. In this review, we examine the knowledge gained on the biology, pathogenesis, immunity and diagnosis of Plasmodium infection from recent proteomic studies. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.

Oxidative stress has been attributed both a key pathogenic and rescuing role in cerebral malaria (CM). In a Plasmodium berghei ANKA murine model of CM, host redox signaling and functioning were examined during the course of neurological damage. Host antioxidant defenses were early altered at the transcriptional level indicated by the gradually diminished expression of superoxide dismutase-1 (sod-1), sod-2, sod-3 and catalase genes. During severe disease, this led to the dysfunctional activity of superoxide dismutase and catalase enzymes in damaged brain regions. Vitagene associated markers (heat shock protein 70 and thioredoxin-1) also showed a decaying expression pattern that paralleled reduced expression of the transcription factors Parkinson disease 7, Forkhead box O 3 and X-box binding protein 1 with a role in preserving brain redox status. However, the oxidative stress markers reactive oxygen/nitrogen species were not accumulated in the brains of CM mice and redox proteomics and immunohistochemistry failed to detect quantitative or qualitative differences in protein carbonylation. Thus, the loss of antioxidant capacity was compensated for in all cerebral regions by progressive upregulation of heme oxygenase-1, and in specific regions by early glutathione peroxidase-1 induction. This study shows for the first time a scenario of cooperative glutathione peroxidase and heme oxygenase-1 upregulation to suppress superoxide dismutase, catalase, heat shock protein-70 and thioredoxin-1 downregulation effects in experimental CM, counteracting oxidative damage and maintaining redox equilibrium. Our findings reconcile the apparent inconsistency between the lack of oxidative metabolite build up and reported protective effect of antioxidant therapy against CM.

The many uses of methylene blue Methylene blue (methylthioninium chloride) is a ‘jack of all trades’ with a litany of clinical uses. In vivo, it is indicated for use as a therapy for drug-induced methemoglobinemia [1-3], can be used for the treatment of infections, pathologies, or poisoning, and as a dye for diagnostics. It is also commonly used as a dye in vitro – for example, as a component in staining of cells, tissues, DNA, parasites, and bacteria [4-6]. In the 1890s, Ehrlich demonstrated its use to target the malarial parasite, and more recently it has been reinvestigated for inclusion in antimalarial regimens in the wake of parasite drug resistance [7-9]. Further examples of its clinical use include treatment of ifosfamide-induced neurotoxicity (although treatment has been reported ineffective), an antidote for cyanide poisoning, visualization of fallopian tubes or ruptured membranes, a marker of tumors, and even as a potential therapy for septic shock and ischemic brain injury [5,8,10-18]. In phase II clinical trials, a modified version of methylene blue is reported to slow cognitive decline in mild–moderate Alzheimer’s disease patients compared to placebo, and phase III trials are planned, although these results remain unpublished [19]. The mechanism of action is unclear – possibly by preventing tau protein aggregation or increasing amyloid-β clearance by enhancing proteasome activities [19,20]. As a photodynamic therapy, methylene blue could be used to treat psoriasis, West Nile virus infection, AIDS-related Kaposi’s sarcoma, antibiotic-resistant bacterial strains and decontaminate blood before transfusion [5,21-25]. Methylene blue has also contributed to drug development, forming the structural chemical basis of other therapeutic drugs, including the antimalarial drug, chloroquine; the antihistamine, promethazine; and the antipsychotic, chlorpromazine [5]. Acknowledging the many uses of methylene blue, this article focuses on the pharmacodynamics of methylene blue in the context of methemoglobinemia treatment and the pathways surrounding this, due to known pharmacogenetic associations that relate to these pathways.

Background and Purpose Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. Experimental Approach Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. Key Results Borrelidin protected against lethal malaria at 0.25 mg·kg−1·day−1. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. Conclusions and Implications Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

* A.R. and M.L. have contributed equally. INTRODUCTION: Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated hematopoietic stem cells. Oxidative stress could play an important role in the pathology of MDS since a correlation has been observed between high ROS levels and decreased survival in MDS patients. In addition, the oxidation of proteins leads to their aggregation, change and conformational changes that can cause the loss of their functions. Due to alteration of the proteome has been described in patients with MDS, in this study we considered the discovery of protein carbonylation to analyze its oxidation pattern. In addition, as the oxidative stress signaling pathways can regulate the cell cycle through p53, we analyzed p21, an important p53 target during the stress response. Finally, the possible benefit of treatment with Deferasirox as antioxidant therapy was studied. METHODS: Carbonylation pattern was analyzed in the different cell lines (n = 14, 6 MDS, 4 reactive controls and 4 MDS + DFX) by immunohistochemistry by derivatization with 2,4-dinitrophenylhydrazine (DNPH) and detection with anti-DNP antibodies. The degree of protein carbonylation was studied, also by derivatization and detection with anti-DNP, in primary cells of erythroblasts expanded for 11 days, by one-dimensional and two-dimensional electrophoresis (n = 16; 7 MDS, 6 controls, 2 reactive controls and 1 MDS + DFX). Expression levels of p21 were analyzed by qRT-PCR (n=19; 8C, 8 MDS, 3DFX). RESULTS: Immunohistochemical assays revealed a higher level of carbonylation in patients with MDS (Fig. 1a). Interestingly, Deferasirox-treated MDS patients had lower levels than non-treated patients (Fig. 1b). In addition, to evaluate the carbonylation in the erythroid precursors, the pattern of carbonylated proteins in the expanded erythroblasts was analyzed. Again, a higher level of MDS was observed compared to the control group, which was reversed after treatment with Deferasirox. Two-dimensional electrophoresis revealed that differences in carbonylation are due to a limited number of proteins or aggregates, rather than to a large spectrum of proteins. Finally, an increased p21 expression was observed in patients with myelodysplasia and, surprisingly, this effect was reversed with Deferasirox treatment. CONCLUSION: Patients with MDS showed a differential protein pattern respect to control patients, both in the myeloid and erythroid series. The increase in oxidative stress in myelodysplastic patients seems to activate signaling pathways involving p21. Treatment with Deferasirox can reverse the level of oxidation and the increase of p21 in patients with MDS. Disclosures Martinez Lopez: Celgene: Research Funding, Speakers Bureau; Jansen: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Resistance to chloroquine (CQ) in Plasmodium falciparum has a major impact on malaria control worldwide. To gain insight into early parasite stress response, mRNA expression profiles were determined for a set of 10 antioxidant defence genes in synchronized CQ-sensitive (3D7) and CQ-resistant (Dd2) clones under transient IC50 CQ-exposure (Dd2, 200 nM; 3D7, 14 nM). Upon 2-h CQ challenge, the mRNA upregulation detected was greater in 3D7 (six genes overexpressed at 1/3 of the intraerythrocytic cycle) than in Dd2 clone (three genes responding), providing evidence of an early transcriptional response to CQ-induced oxidative stress which might underlie some of the parasite's metabolic adaptation to the drug.

In cerebral malaria, the most severe complication of malaria, both neurotransmission mechanisms and energy metabolism are affected. To understand how metabolic changes modify neurotransmission, we examined P2 receptor expression in a murine model of cerebral malaria. Quantitative polymerase chain reaction experiments revealed that parasite deposition was greatest in the cerebellum, compared with other areas of the brain, suggesting a correlation between brain parasitemia and loss of control of movement. Infected mice showed modified patterns of expression of P2 receptor subtype messenger RNA (mRNA), depending on both the specific purinergic receptor and the cerebral region analyzed. Immunohistochemical studies indicated altered levels of protein expression by these receptors in infected brains and, in some cases, a pattern of expression different from that noted in control mice. These differences in both the amount of mRNA and the protein distribution of P2 receptors observed in the different brain sites in infected mice suggest an important role for P2 receptors in either provoking cerebral damage or conferring neuroprotection.

Resistance of Plasmodium falciparum to chloroquine hinders malaria control in endemic areas. Current hypotheses on the action mechanism of chloroquine evoke its ultimate interference with the parasite's oxidative defence systems. Through carbonyl derivatization by 2,4-dinitrophenylhydrazine and proteomics, we compared oxidatively modified proteins across the parasite's intraerythrocytic stages in untreated and transiently IC 50 chloroquine-treated cultures of the chloroquine-resistant P. falciparum strain Dd2. Functional plasmodial protein groups found to be most oxidatively damaged were among those central to the parasite's physiological processes, including protein folding, proteolysis, energy metabolism, signal transduction, and pathogenesis. While an almost constant number of oxidized proteins was detected across the P. falciparum life cycle, chloroquine treatment led to increases in both the extent of protein oxidation and the number of proteins oxidized as the intraerythrocytic cycle progressed to mature stages. Our data provide new insights into early molecular effects produced by chloroquine in the parasite, as well as into the normal protein-oxidation modifications along the parasite cycle. Oxidized proteins involved in the particular parasite drug-response suggest that chloroquine causes specific oxidative stress, sharing common features with eukaryotic cells. Targeting these processes might provide ways of combating chloroquine-resistance and developing new antimalarial drugs.

Babesiosis is an emerging zoonosis now found in several areas of the world. Using PCR and indirect immunofluorescence assay, we have diagnosed the first case of human babesiosis caused by Babesia microti in Spain. Diagnosis was delayed because of the nonspecific clinical symptoms that occurred in an immunocompetent patient. We thank Cesar Eguiluz for his contribution to this study. This work is funded by the Surveillance Program of the Centro Nacional de Microbiología, the Center for Collaborative Research (RETIC-RICET), and the grants from Ministerio de Economia y Competitividad of Spain (grant numbers AGL2010-21774 and AGL2014-56193-R awarded to E.M. and L.M.G. and grant number BIO2013-44565-R awarded to J.M.B.) and the NIH of the USA (grant numbers HL105694 and HL129215 awarded to C.A.L.).

The influence of feeding system on growth performance, carcass characteristics, and fatty acids profile of subcutaneous backfat and lipogenic enzymes activity was studied. The following feeding systems were applied: HH: group of 12 pigs fed with a high feeding level during growth and fattening periods; HL: group of 12 pigs fed with a high feeding level during growth period and a low feeding level during fattening period; LH: group of 12 pigs fed with a low feeding level during growth period and a high feeding level during fattening period; LL: group of 12 pigs fed with a low feeding system during growth and fattening periods. The high and low feeding levels applied during growth and fattening periods were 70 and 35 g feed/kg live weight 0.75 (LW 0.75 ) day and 121 and 87 g feed/kg LW 0.75 day, respectively. The duration of the growth period was 163 days in all groups. The ages at slaughter of HH, HL, LH and LL pigs were approximately 383, 406, 439 and 503 days, respectively. The HH and LL pigs had the higher and lower average daily gain respectively, while the LH pigs presented the best feed conversion ratio during fattening period. At slaughter, the LL pigs had the higher value of loin width and the lower values of backfat thicknesses and Longissimus dorsi muscle intramuscular fat percentage (P n -9 and ΣMUFA (monounsaturated) acids than those from HH and HL pigs. The subcutaneous backfat inner layer from LL pigs had a lower C16:0 and ΣSFA (saturated) acids proportions than those from remaining groups of pigs. The activity of the fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PD) enzymes, measured in subcutaneous backfat, at the end of growing period was significantly (P

Deficiency of glucose-6-phosphate dehydrogenase is a very common X-linked genetic disorder though most deficient people are asymptomatic. A number of different G6PD variants have reached polymorphic frequencies in different parts of the world due to the relative protection they confer against malaria infection. People, usually males, with deficient alleles are susceptible to neonatal jaundice, and acute hemolytic anemia, usually during infection, after treatment with certain drugs or after eating fava beans. Very rarely de novo mutations can arise causing the more severe condition of chronic nonspherocytic hemolytic anemia. Altogether 160 different mutations have been described. The majority of mutations cause red cell enzyme deficiency by decreasing enzyme stability. The polymorphic mutations affect amino acid residues throughout the enzyme and decrease the stability of the enzyme in the red cell, possibly by disturbing protein folding. The severe mutations mostly affect residues at the dimer interface or those that interact with a structural NADP molecule that stabilizes the enzyme.

To maximize growth, farmed fish are fed high-fat diets, which can lead to high tissue lipid concentrations that have an impact on quality. The intake of conjugated linoleic acid (CLA) reduces body fat in mammals and this study was undertaken to determine the effects of dietary CLA on growth, composition, and postprandial metabolic variables in sea bream. Fish were fed 3 diets containing 48 g/100 g protein and 24 g/100 g fat, including fish oil supplemented with 0 (control), 2, or 4% CLA for 12 wk. Feed intake, specific growth rate, total body fat, and circulating somatolactin concentration were lower in fish fed CLA than in controls. Feed efficiency was greater in fish fed 2% CLA than in controls. Liver triglyceride concentrations were higher in fish fed 4% CLA and muscle triglyceride concentrations were lower in fish fed both CLA diets than in controls. Hepatic fatty acyl desaturase and elongase mRNA levels in fish fed CLA were lower than in controls. Metabolic differences between controls and CLA-fed fish were observed at 6 h but not at 24 h after the last meal, including lower postprandial circulating triglyceride concentrations, higher hepatic acyl-CoA-oxidase, and lower L-3-hydroxyacyl-CoA dehydrogenase activities in CLA-fed fish than in controls. Dietary CLA did not affect enzymes involved in lipogenesis including hepatic fatty acid synthase and malic enzyme, but it decreased glucose 6-phosphate dehydrogenase activity at 24 h, but not at 6 h after feeding. The data suggest that CLA intake in sea bream has little effect on hepatic lipogenesis, channels dietary lipid from adipose tissue to the liver, and switches hepatic mitochondrial to peroxisomal beta-oxidation.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethalPlasmodium yoelii yoelii17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses ofP. yoelii yoelii17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites.

An experiment was conducted to study dietary fat type (fish oil (FO) vs. vegetable oil) effect on lipid and glucose metabolism in post-smolt Atlantic salmon. Duplicate groups of salmon were fed one of eight diets in which the two fat sources FO (long chain n-3 fatty acids, FA) or linseed oil (LO) (short chain n-3 FA) were combined in a 2 × 4 factorial design with sunflower oil (SO) (rich in n-6 FA) at inclusion levels of 0, 25, 50 and 75% of total added fat. The effects of the diets on plasma metabolites, the activity of selected enzymes involved in lipid metabolism, biometric indices and muscle glucose transporter GLUT4 expression were determined after 12 weeks of feeding. Lower viscero-somatic indices (VSI) and fatty livers were observed in fish fed LO based diets. Increasing inclusion levels of SO affected plasma glucose concentration in fish fed FO based diets, and plasma triglycerides, which decreased in a linear and quadratic pattern in fish fed FO based diets, but increased linearly in fish fed LO based diets. Specific activity of liver carnitine palmitoyl transferase I (CPT I) and glucose-6-phosphate dehydrogenase (G6PD) and plasma nonesterified fatty acids (NEFA) concentration was higher in fish fed LO based diets. Two GLUT4 isoforms I and II have been described in muscle and proved to be differentially expressed related to dietary fatty acids. In summary, dietary fat type affects lipid metabolism in post-smolted Atlantic salmon. In addition, a possibility to interfere on glucose metabolism by means of dietary fat type is discussed.

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity ofor=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

BACKGROUND AND OBJECTIVES Fluorescent molecular beacons have been employed as hybridization probes in real time quantitative PCR to quantify residual disease in multiple myeloma (MM). Design and methods After clinical diagnosis of MM, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. A molecular beacon probe for the beta-globin gene was used as a reference control to calculate relative amounts of the clonal B-cell population. Results Optimization of probe design resulted in the use of a competitive sequence at the IgH area target between the loop and part of the stem of the molecular beacon. Cycling conditions and fluorescence temperature acquisition were optimized for a Light Cycler. To validate this method for the follow-up of treated MM patients, we investigated accuracy, as well as interassay and intrassay reproducibility. Conclusions Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM.

Piracanjuba (Brycon orbignyanus) is a Brazilian migratory fast-growing omnivore, very appreciated as a sport fish, which is threatened to extinction in Southern Brazil due to stock over exploitation and dam building. Therefore, efforts have been made to raise this fish in captivity for reintroduction and aquaculture purposes. In the present study, the effects of different dietary protein and lipid concentrations on piracanjub a fingerlings growth performance, feed utilization, body composition, hepatosomatic index (HSI) and activity of the lipogenic enzymes fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) were investigated usin ga2◊3f actorial experiment. Six casein‐gelatin based diets were prepared combining two protein (30% and 32%) and three lipid concentrations (5.5%, 8.8% and 12.1%). Eleven fish, average weight 11.30 ± 0.1 g, were held in each of 18 100‐l aquaria, supplied with recirculating freshwater. Each diet was randomly assigned to triplicate groups of fish and fed to apparent satiation, twice a day for 100 d. Piracanjuba fingerlings’ daily weight gain (0.36‐0.40 g), specific growth rate (1.43‐1.51%), feed utilization and HSI were not influenced by dietary protein or lipid concentration. However, body composition was directly affected by dietary treatment. An increase in body fat and dry matter was observed as dietary lipid increased, for both dietary protein concentrations tested. The activity of FAS was depressed by increasing dietary fat levels but the G6PD activity did not differ among dietary treatments, although ME activity showed some regulation by dietary protein. These results indicate that an increase from 5.5% to 12.1% in the dietary lipid, at a dietary protein concentration of 30% or 32%, promotes body fat accumulation in piracanjuba fingerlings with no improvement in growth, suggesting that the lipid requirement for this species should be 5% or less, when raised for commercial purposes. However, the additional energy reserve from body fat accumulation could be desirable for piracanjuba fingerlings produced for stock enhancement.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

ICR mice have heterogeneous susceptibility to lethal Plasmodium yoelii yoelii 17XL from the first days of experimental infection as evidenced by the different parasitemia levels and clinical outcomes. This mouse model has revealed specific immune responses on peripheral blood correlating with the infection fate of the animals. To search for immune-markers linked to parasitemia we examined B lymphocytes in organs of the immune system as key effectors of rodent immunity against malaria. To determine changes in immune cellularity fostered by the different prognostic parasitemia we examined B cell subsets in low (15%) and high (50%) parasitized mice during the first days of the infection. In the case of surviving mice, we studied the preservation of memory immune response 500 days after the primary P. yoelii challenge. Correlating with the parasitemia level, it was observed an increase in total cellularity of spleen during the first week of infection which remained after 16 months of the infection in surviving animals. B cell subsets were also modified across the different infection fates. Subpopulation as follicular B cells and B-1 cells proportions behaved differently depending on the parasitemia kinetics. In addition, peritoneal cavity cells proliferated in response to high parasitemia. More significantly, P. yoelii -specific memory B cells remained in the spleen 500 days after the primo-infection. This study demonstrates that B cell kinetics is influenced by the different parasitemia courses which are naturally developed within a same strain of untreated mice. We show that high levels of parasitemia at the beginning of infection promote an extremely fast and exacerbate response of several cell populations in spleen and peritoneal cavity that, in addition, do not follow the kinetics observed in peripheral blood. Furthermore, our results describe the longest persistence of memory B cells long time upon a single malaria infection in mice.

We evaluated the effects of dietary fat type on fat metabolism and deposition in broiler chickens. Birds were fed diets containing either 8 g dietary saturated (beef tallow) or polyunsaturated fat (sunflower oil)/100 g for 32 d. The abdominal fat deposition of chickens fed the sunflower oil-enriched diet was significantly lower than that of chickens fed the tallow-enriched diet (2.63 +/- 0.47 versus 3.03 +/- 0.44 g/100 g live wt.; P = 0.033). The specific activities of heart carnitine palmitoyltransferase I and L-3-hydroxyacyl-CoA dehydrogenase were higher (P < or = 0.03) in chickens fed the sunflower oil-enriched diets, indicating a greater rate of beta-oxidation. Liver fatty acid synthetase activity was lower (P = 0.01) in chickens fed the sunflower oil-enriched diet, suggesting reduced hepatic lipogenesis in this group. Postprandial plasma triglyceride levels were significantly lower (P < 0.05) in birds fed the sunflower oil-enriched diet, indicating a higher rate of dietary lipid clearance from the bloodstream to tissues. In conclusion, the lower fat deposition observed in broilers fed sunflower oil-enriched diets appears to be the net result of an increased rate of lipid catabolism and lower rate of fatty acid synthesis despite higher dietary fat absorption.

We analyzed glucose-6-phosphate dehydrogenase, the rate-controlling enzyme of the pentose phosphate pathway and free sulfhydryls, to study redox balance in Alzheimer disease. Glucose-6-phosphate dehydrogenase plays a pivotal role in homeostatic redox control by providing reducing equivalents to glutathione, the major nonenzymatic cellular antioxidant. There is a multitude of evidence that marks oxidative stress proximally in the natural history of Alzheimer disease. Consistent with a role for glutathione in defense against increased reactive oxygen, we found an upregulation of glucose-6-phosphate dehydrogenase together with increased sulfhydryls in Alzheimer disease. These data indicate that reductive compensation may play an important role in combating oxidative stress in Alzheimer disease.

A growth trial was conducted with groups of European seabass having an initial weight of 6 g to study the lipogenic action of dietary protein and non-protein energy supplies. Six experimental diets were formulated to contain one of two crude protein levels (43 and 52%) with digestible protein (DP) to digestible energy (DE) ratios ranging from 19 to 26 mg/kJ. At the end of the growth trial (12 weeks), the activities of liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), malic enzyme (ME, EC 1.1.1.40), ATP citrate lyase (ACL, EC 4.1.3.8), acetyl-CoA carboxylase (ACoAC, EC 6.4.1.2) and fatty acid synthetase (FAS, EC 2.3.1.38) were measured. Digestibility of main dietary components was also determined over a three-week period. At each protein level, an increase in dietary DE led to improved growth performance, protein efficiency, daily N deposition and to a reduction of N loss. Best results were achieved at 40% DP and a DP/DE ratio of 19–20 mg/kJ. G6PD, ME and ACoAC were found to be the key regulatory enzymes in the lipogenic pathway, with G6PD being the main NADPH-generating enzyme. Activities of G6PD, ME, ACL and FAS were reduced with increasing fat intake. Activities of G6PD, ME and ACL were increased with increasing starch intake. ACoAC activity was negatively correlated with starch intake and positively with fat intake.

Malaria infection in humans elicits a wide range of immune responses that can be detected in peripheral blood, but we lack detailed long-term follow-up data on the primary and subsequent infections that lead to naturally acquired immunity. Studies on antimalarial immune responses in mice have been based on models yielding homogenous infection profiles. Here, we present a mouse model in which a heterogeneous course of Plasmodium yoelii lethal malaria infection is produced in a non-congenic ICR strain to allow comparison among different immunological and clinical outcomes. Three different disease courses were observed ranging from a fatal outcome, either early or late, to a self-resolved infection that conferred long-term immunity against re-infection. Qualitative and quantitative changes produced in leukocyte subpopulations and cytokine profiles detected in peripheral blood during the first week of infection revealed that monocytes, dendritic cells and immature B cells were the main cell subsets present in highly-parasitized mice dying in the first week after infection. Besides, CD4(+)CD25(high) T cells expanded at an earlier time point in early deceased mice than in surviving mice and expressed higher levels of intracellular Foxp3 protein. In contrast, survivors showed a limited increase of cytokines release and stable circulating innate cells. From the second week of infection, mice that would die or survive showed similar immune profiles, although CD4(+)CD25(high) T cells number increased earlier in mice with the worst prognosis. In surviving mice the expansion of activated circulating T cell and switched-class B cells with a long-term protective humoral response from the second infection week is remarkable. Our results demonstrate that the follow-up studies of immunological blood parameters during a malaria infection can offer information about the course of the pathological process and the immune response.

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum ( p.f. -ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f. -ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f. -ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f. -ChoK as an accessible drug target against the parasite.

The role of neurotrophic factors on the integrity of the central nervous system (CNS) during cerebral malaria (CM) infection remains obscure, but the long-standing neurocognitive sequelae often observed in rescued children can be attributed in part to the modulation of neuronal survival and synaptic plasticity. To discriminate the contribution of key responses in the time-sequence of the pathogenic events that trigger the development of neurocognitive malaria syndrome we defined four stages (I-IV) of the neurological progression of CM in C57BL/6 mice infected with Plasmodium berghei ANKA. Upregulation of ICAM-1, VCAM-1, e-selectin and p-selectin expression was detected in all cerebral regions before parasitized red blood cells (pRBC) accumulation. As the severity of symptoms increased, BDNF mRNA progressively diminished in several brain regions, earliest in the thalamus-hypothalamus, cerebellum, brainstem and cortex, and correlated with a four-stage disease sequence. Immunohistochemical confocal microscopy revealed changes in the BDNF distribution pattern, suggesting altered axonal transport. During CM progression, molecular markers of neurological infection and inflammation in the parasite and the host, respectively, were accompanied by a switch in the brain constitutive proteasome to the immunoproteasome, which could impede normal protein turnover. In parallel with BDNF downregulation, NCAM expression also diminished with increased CM severity. Together, these data suggest that changes in BDNF availability could be involved in the pathogenesis of CM.

We report a case of an African patient with sickle cell trait who was diagnosed in Spain with B-cell lymphoma. Blood smears were negative for malaria, and no plasmodium antigens were detected in the blood. To treat his lymphoma, the patient underwent chemotherapy and autologous stem cell transplantation. Following a splenectomy due to a worsening condition, he developed clinical malaria with detectable parasitemia. This case suggests that the humoral response and parasite removal by the spleen may afford protection from overt disease and may even help maintain subclinical human reservoirs of the disease.

In this issue of Blood, Makarona et al demonstrate that histone deacetylase (HDAC) inhibitors (HDACis) in glucose-6-phosphate dehydrogenase (G6PD)-deficient cells reinstates enzyme activity by boosting gene transcription.1 This therapeutic approach opens new avenues for preclinical and clinical studies to treat not only chronic nonspherocytic hemolytic anemia caused by severe G6PD variants,2 but also other genetic diseases.

Multiple glucose-6-phosphate dehydrogenase (G6PD)-deficient alleles have reached polymorphic frequencies because of the protection they confer against malaria infection. A protection mechanism based on enhanced phagocytosis of parasitized G6PD-deficient erythrocytes that are oxidatively damaged is well accepted. Although an association of this phenotype with the impairment of the antioxidant defense in G6PD deficiency has been demonstrated, the dysfunctional pathway leading to membrane damage and modified exposure of the malaria-infected red cell to the host is not known. Thus, in this study, erythrocytes from the common African variant G6PD A- were used to analyze by redox proteomics the major oxidative changes occurring in the host membrane proteins during the intraerythrocytic development of Plasmodium falciparum, the most lethal malaria parasite. Fifteen carbonylated membrane proteins exclusively identified in infected G6PD A- red blood cells revealed selective oxidation of host proteins upon malarial infection. As a result, three pathways in the host erythrocyte were oxidatively damaged in G6PD A-: (1) traffic/assembly of exported parasite proteins in red cell cytoskeleton and surface, (2) oxidative stress defense proteins, and (3) stress response proteins. Additional identification of hemichromes associated with membrane proteins also supports a role for specific oxidative modifications in protection against malaria by G6PD polymorphisms.

Proteins modified by 4-hydroxy-2-nonenal (HNE) are cellular markers of oxidative stress in health and disease. HNE is generated by free radical chain reactions during oxidative stress as a major end-product of the oxidative fatty acid metabolism. Identification and quantitative analysis of HNE-modified proteins are readily performed by using specific antibodies raised against them. Further on, the identification of the amino acid residues involved in the HNE-modification is an additional step in proteomic post-transcriptional modification analysis to explain the nature of the specificity underlying oxidative stress mechanisms. For this purpose, a combined protocol of immune-detection, peptide enrichment, mass spectrometry, and de novo protein sequencing has been developed. The methodology was first examined in the model protein bovine serum albumin (BSA), allowing the comparison of matrix-assisted laser desorption/ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) performance and sensitivity. Peptide enrichment was optimized by affinity chromatography on HNE-BSA resulting in increased sensitivity. Identification of amino acid residues modified by HNE was finally ascertained by de novo sequencing analysis. The improved methodology was demonstrated on human erythrocyte membrane proteins allowing the identification of HNE-lysine and HNE-histidine Michael adducts in the β-spectrin under physiological conditions.

The high prevalence of glucose 6-phosphate dehydrogenase (G6PD) deficiency in African populations is due almost entirely to the enzyme variant A-, which differs from the wild-type G6PD B by two amino acid replacements, 68 Val-->Met and 126 Asn-->Asp. The non-deficient polymorphic variant G6PD A contains only the mutation 126 Asn-->Asp. The frequencies of the G6PD A and of the G6PD A- genes in parts of Africa are both about 0.2. The 68 Val-->Met mutation has not been found in a B background. This could be because the 68 Val-->Met mutation happened to arise in an A gene in the first instance, or because the 68 Val-->Met mutation alone is not sufficient to cause G6PD deficiency. We have approached this question by producing G6PD B, A, A-, and G6PD 68 Val-->Met in a bacterial expression system and analysing their biochemical properties. With each single mutation we found a slight decrease in both the specific activity and the yield of enzyme when compared to G6PD B. When both mutations were introduced together, there was a roughly additive effect on specific activity, but a much more drastic effect on enzyme yield (4% of normal). This synergistic effect was also demonstrated on thermal stability, especially at low NADP concentrations. Comparable results were produced when the replacement 119 Gln-->Glu was studied instead of 126 Asn-->Asp. We infer that the coexistence of the two mutations is responsible for enzyme deficiency in G6PD A- because they act synergistically in causing instability of the enzyme.

This protocol describes a method for preparing cultures of Plasmodium falciparum synchronized at any intraerythrocytic stage. Using this method, around 60% parasitized cells may be obtained. On the basis of Trager and Jensen's original continuous culture method, our approach relies on the use of fresh human blood not older than 2 weeks, a low hematocrit between 0.8 and 1.5%, a starting frozen inoculum of 10% ring-stage parasitemia, human serum replaced with AlbuMAX I and alternating sorbitol and Percoll synchronization methods to shorten the cycle window to 4–6 h and reduce sorbitol toxicity. From our synchronized high parasite density cultures, 3–5 ml of infected red blood cells can be obtained in 1 week, corresponding to 1.2 mg of total parasite protein per ml of harvested culture. On the basis of the variables parasitemia and packed cell volume, we provide an equation to accurately calculate the amount of complete medium required every 24 h corrected for the cycle stage and capacity of the culture flask. Ten days suffice to complete the protocol from a frozen stock of parasites.