Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC-MS/MS from Dried Serum Spots.

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

Immune checkpoints and cancer immunotherapies: insights into newly potential receptors and ligands.

Checkpoint markers and immune checkpoint inhibitors have been increasingly identified and developed as potential immunotherapeutic targets in various human cancers. Despite valuable efforts to discover novel immune checkpoints and their ligands, the precise roles of their therapeutic functions, as well as the broad identification of their counterpart receptors, remain to be addressed. In this context, it has been suggested that various putative checkpoint receptors can be induced upon activation. In the tumor microenvironment, T cells, as crucial immune response against malignant diseases as well as other immune central effector cells, such as natural killer cells, are regulated via co-stimulatory or co-inhibitory signals from immune or tumor cells. Studies have shown that exposure of T cells to tumor antigens upregulates the expression of inhibitory checkpoint receptors, leading to T-cell dysfunction or exhaustion. Although targeting immune checkpoint regulators has shown relative clinical efficacy in some tumor types, most trials in the field of cancer immunotherapies have revealed unsatisfactory results due to de novo or adaptive resistance in cancer patients. To overcome these obstacles, combinational therapies with newly discovered inhibitory molecules or combined blockage of several checkpoints provide a rationale for further research. Moreover, precise identification of their receptors counterparts at crucial checkpoints is likely to promise effective therapies. In this review, we examine the prospects for the application of newly emerging checkpoints, such as T-cell immunoglobulin and mucin domain 3, lymphocyte activation gene-3, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), V-domain Ig suppressor of T-cell activation (VISTA), new B7 family proteins, and B- and T-cell lymphocyte attenuator, in association with immunotherapy of malignancies. In addition, their clinical and biological significance is discussed, including their expression in various human cancers, along with their roles in T-cell-mediated immune responses.

Co-Occurrence of G6PD Deficiency and SCT among Pregnant Women Exposed to Infectious Diseases.

During pregnancy, women have an increased relative risk of exposure to infectious diseases. This study was designed to assess the prevalence of the co-occurrence of glucose-6-phosphate dehydrogenase deficiency (G6PDd) and sickle cell trait (SCT) and the impact on anemia outcomes among pregnant women exposed to frequent infectious diseases. Over a six-year period (March 2013 to October 2019), 8473 pregnant women attending antenatal clinics (ANCs) at major referral hospitals in Northern Ghana were recruited and diagnosed for common infectious diseases (malaria, syphilis, hepatitis B, and HIV), G6PDd, and SCT. The prevalence of all the infections and anemia did not differ between women with and without G6PDd (χ2 < 3.6, p > 0.05 for all comparisons). Regression analysis revealed a significantly higher proportion of SCT in pregnant women with G6PDd than those without G6PDd (AOR = 1.58; p < 0.011). The interaction between malaria and SCT was observed to be associated with anemia outcomes among the G6PDd women (F-statistic = 10.9, p < 0.001). Our findings show that anemia is a common condition among G6PDd women attending ANCs in northern Ghana, and its outcome is impacted by malaria and SCT. This warrants further studies to understand the impact of antimalarial treatment and the blood transfusion outcomes in G6PDd/SCT pregnant women.

Isolation and Characterization of NpCI, a New Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita peloronta with Anti-Plasmodium falciparum Activity.

Metallocarboxypeptidases are zinc-dependent peptide-hydrolysing enzymes involved in several important physiological and pathological processes. They have been a target of growing interest in the search for natural or synthetic compound binders with biomedical and drug discovery purposes, i.e., with potential as antimicrobials or antiparasitics. Given that marine resources are an extraordinary source of bioactive molecules, we screened marine invertebrates for new inhibitory compounds with such capabilities. In this work, we report the isolation and molecular and functional characterization of NpCI, a novel strong metallocarboxypeptidase inhibitor from the marine snail Nerita peloronta. NpCI was purified until homogeneity using a combination of affinity chromatography and RP-HPLC. It appeared as a 5921.557 Da protein with 53 residues and six disulphide-linked cysteines, displaying a high sequence similarity with NvCI, a carboxypeptidase inhibitor isolated from Nerita versicolor, a mollusc of the same genus. The purified inhibitor was determined to be a slow- and tight-binding inhibitor of bovine CPA (Ki = 1.1·× 10-8 mol/L) and porcine CPB (Ki = 8.15·× 10-8 mol/L) and was not able to inhibit proteases from other mechanistic classes. Importantly, this inhibitor showed antiplasmodial activity against Plasmodium falciparum in an in vitro culture (IC50 = 5.5 µmol/L), reducing parasitaemia mainly by inhibiting the later stages of the parasite's intraerythrocytic cycle whilst having no cytotoxic effects on human fibroblasts. Interestingly, initial attempts with other related proteinaceous carboxypeptidase inhibitors also displayed similar antiplasmodial effects. Coincidentally, in recent years, a metallocarboxypeptidase named PfNna1, which is expressed in the schizont phase during the late intraerythrocytic stage of the parasite's life cycle, has been described. Given that NpCI showed a specific parasiticidal effect on P. falciparum, eliciting pyknotic/dead parasites, our results suggest that this and related inhibitors could be promising starting agents or lead compounds for antimalarial drug discovery strategies.

Protein Susceptibility to Peroxidation by 4-Hydroxynonenal in Hereditary Hemochromatosis.

Iron overload caused by hereditary hemochromatosis (HH) increases free reactive oxygen species that, in turn, induce lipid peroxidation. Its 4-hydroxynonenal (HNE) by-product is a well-established marker of lipid peroxidation since it reacts with accessible proteins with deleterious consequences. Indeed, elevated levels of HNE are often detected in a wide variety of human diseases related to oxidative stress. Here, we evaluated HNE-modified proteins in the membrane of erythrocytes from HH patients and in organs of Hfe-/- male and female mice, a mouse model of HH. For this purpose, we used one- and two-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF analysis. We identified cytoskeletal membrane proteins and membrane receptors of erythrocytes bound to HNE exclusively in HH patients. Furthermore, kidney and brain of Hfe-/- mice contained more HNE-adducted protein than healthy controls. Our results identified main HNE-modified proteins suggesting that HH favours preferred protein targets for oxidation by HNE.

Microscopic and submicroscopic infection by Plasmodium falciparum: Immunoglobulin M and A profiles as markers of intensity and exposure.

Assessment of serological Plasmodium falciparum-specific antibodies in highly endemic areas provides valuable information about malaria status and parasite exposure in the population. Although serological evidence of Plasmodium exposure is commonly determined by Plasmodium-specific immunoglobulin G (IgG) levels; IgM and IgA are likely markers of malaria status that remain relatively unexplored. Previous studies on IgM and IgA responses have been based on their affinity for single antigens with shortage of immune responses analysis against the whole Plasmodium proteome. Here, we provide evidence of how P. falciparum infection triggers the production of specific IgM and IgA in plasma and its relationship with parasite density and changes in hematological parameters. A total of 201 individuals attending a hospital in Breman Asikuma, Ghana, were recruited into this study. Total and P. falciparum-specific IgM, IgA, and IgG were assessed by ELISA and examined in relation to age (0-5, 14-49, and ≥50 age ranges); infection (submicroscopic vs. microscopic malaria); pregnancy and hematological parameters. Well-known IgG response was used as baseline control. P. falciparum-specific IgM and IgA levels increased in the population with the age, similarly to IgG. These data confirm that acquired humoral immunity develops by repeated infections through the years endorsing IgM and IgA as exposure markers in endemic malaria regions. High levels of specific IgA and IgM in children were associated with microscopic malaria and worse prognosis, because most of them showed severe anemia. This new finding shows that IgM and IgA may be used as diagnostic markers in this age group. We also found an extremely high prevalence of submicroscopic malaria (46.27% on average) accompanied by IgM and IgA levels indistinguishable from those of uninfected individuals. These data, together with the observed lack of sensitivity of rapid diagnostic tests (RDTs) compared to PCR, invoke the urgent need to implement diagnostic markers for submicroscopic malaria. Overall, this study opens the potential use of P. falciparum-specific IgM and IgA as new serological markers to predict malaria status in children and parasite exposure in endemic populations. The difficulties in finding markers of submicroscopic malaria are highlighted, emphasizing the need to explore this field in depth.

Hypothyroidism confers tolerance to cerebral malaria.

The modulation of the host's metabolism to protect tissue from damage induces tolerance to infections increasing survival. Here, we examined the role of the thyroid hormones, key metabolic regulators, in the outcome of malaria. Hypothyroidism confers protection to experimental cerebral malaria by a disease tolerance mechanism. Hypothyroid mice display increased survival after infection with Plasmodium berghei ANKA, diminishing intracranial pressure and brain damage, without altering pathogen burden, blood-brain barrier disruption, or immune cell infiltration. This protection is reversed by treatment with a Sirtuin 1 inhibitor, while treatment of euthyroid mice with a Sirtuin 1 activator induces tolerance and reduces intracranial pressure and lethality. This indicates that thyroid hormones and Sirtuin 1 are previously unknown targets for cerebral malaria treatment, a major killer of children in endemic malaria areas.

In vitro antiplasmodial activity of selected plants from the Colombian North Coast with low cytotoxicity.

Background: Plants are an important option in the treatment of malaria, especially in endemic regions, and are a less expensive and more accessible alternative with a lower risk of toxicity. Colombia has a great diversity of plants, and evaluation of natural extracts could result in the discovery of new compounds for the development of antimalarial drugs. The purpose of this work was to evaluate the in vitro antiplasmodial activity and the cytotoxicity of plant extracts from the Colombian North Coast against Plasmodium falciparum. Materials and Methods: The antiplasmodial activity of 12 plant species from the Colombian North Coast that are used in traditional medicine was evaluated through in vitro cultures of P. falciparum, and the cytotoxicity of extracts of these species to human cells was determined. Plant extracts with high antiplasmodial activity were subjected to preliminary phytochemical screening. Results: Extracts from five plants had promising antiplasmodial activity. Specifically, Bursera simaruba (Burseraceae) (bark), Guazuma ulmifolia Lam. (Malvaceae) (whole plant), Murraya exotica L. (Rutaceae) (leaves), Hippomane mancinella L. (Euphorbiaceae) (seeds), and Capparis odoratissima Jacq. (Capparaceae) (leaves). Extracts presented 50% inhibitory concentration values between 1 and 9 µg/ml. Compared to no extract, these active plant extracts did not show cytotoxic effects on mononuclear cells or hemolytic activity in healthy human erythrocytes. Conclusions: The results obtained from this in vitro study of antiplasmodial activity suggest that active plant extracts from the Colombian North Coast are promising for future bioassay-guided fractionation to allow the isolation of active compounds and to elucidate their mechanism of action against Plasmodium spp.

Home Sweet Home: Plasmodium vivax-Infected Reticulocytes-The Younger the Better?

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.

The Potential Role of Pro-Inflammatory and Anti-Inflammatory Cytokines in Epilepsy Pathogenesis.

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)-1ß, 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-ß), IL-8, IL-12, IL-18, and macrophage inflammatory protein (MIP) as potential proinflammatory mediators in the neuropathology of epilepsy.

Plasmodium falciparum immunodominant IgG epitopes in subclinical malaria.

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-stage Plasmodium falciparum rapidly 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 for P. falciparum antigens and to identify epitopes associated with exposure and clinical disease. Sera from malaria cases identified five prevalent antigens recognized by all analyzed patients' IgGs. Epitope mapping of them, using adult and children sera samples from an endemic malaria region in Ghana segregated into patients with positive or negative subclinical detection of P. falciparum, revealed binding specificity for two 20-mer immunodominant antigenic regions within the START-related lipid transfer protein and the protein disulfide isomerase PDI8. These 20-mer epitopes 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 humoral response against START and PDI8 antigens may be triggered at submicroscopic parasitemia levels in children and may eventually be used to differentially diagnose subclinical malaria in children.

Protein Carbonylation in Patients with Myelodysplastic Syndrome: An Opportunity for Deferasirox Therapy.

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.

Correction: Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

[This corrects the article DOI: 10.1371/journal.pone.0219780.].

Recombinant rabbit beta nerve growth factor production and its biological effects on sperm and ovulation in rabbits.

In some induced-ovulating species, beta nerve growth factor (ß-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant ß-NGF from rabbit tissue (rrß-NGF) to address the following objectives: 1) to compare rabbit ß-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrß-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrß-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 rß-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 ß-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 rrß-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 rrß-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 rrß-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed.

Screening for retroviruses and hepatitis viruses using dried blood spots reveals a high prevalence of occult hepatitis B in Ghana.

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.

A role for Th1-like Th17 cells in the pathogenesis of inflammatory and autoimmune disorders.

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.

Characterization of ß-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between ß-NGF and testosterone levels with seminal quality during sexual maturation.

ß-Nerve Growth Factor (ß-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 ß-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. ß-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 ß-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate ß-NGF concentration, and testosterone levels were also assessed in BP. The highest ß-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual ß-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 ß-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, ß-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. ß-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 ß-NGF.

Gene expression and immunolocalization of low-affinity neurotrophin receptor (p75) in rabbit male reproductive tract during sexual maturation.

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.

First homology model of Plasmodium falciparum glucose-6-phosphate dehydrogenase: Discovery of selective substrate analog-based inhibitors as novel antimalarial agents.

In Plasmodium falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase‒6-phosphogluconolactonase (PfG6PD‒6PGL) is involved in the catalysis of the first reaction of the pentose phosphate pathway. Since this enzyme has a key role in parasite development, its unique structure represents a potential target for the discovery of antimalarial drugs. Here we describe the first 3D structural model of the G6PD domain of PfG6PD‒6PGL. Compared to the human enzyme (hG6PD), the 3D model has enabled the identification of a key difference in the substrate-binding site, which involves the replacement of Arg365 in hG6PD by Asp750 in PfG6PD. In a prospective validation of the model, this critical change has been exploited to rationally design a novel family of substrate analog-based inhibitors that can display the necessary selectivity towards PfG6PD. A series of glucose derivatives featuring an α-methoxy group at the anomeric position and different side chains at position 6 bearing distinct basic functionalities has been synthesized, and their PfG6PD and hG6PD inhibitory activities and their toxicity against parasite and mammalian cells have been assessed. Several compounds displayed micromolar affinity (Ki up to 23 µM), favorable selectivity (up to > 26-fold), and low cytotoxicity. Phenotypic assays with P. falciparum cultures revealed high micromolar IC50 values, likely as a result of poor internalization of the compounds in the parasite cell. Overall, these results endorse confidence to the 3D model of PfG6PD, paving the way for the use of target-based drug design approaches in antimalarial drug discovery studies around this promising target.

Plasmodium species differentiation by non-expert on-line volunteers for remote malaria field diagnosis.

BACKGROUND: Routine field diagnosis of malaria is a considerable challenge in rural and low resources endemic areas mainly due to lack of personnel, training and sample processing capacity. In addition, differential diagnosis of Plasmodium species has a high level of misdiagnosis. Real time remote microscopical diagnosis through on-line crowdsourcing platforms could be converted into an agile network to support diagnosis-based treatment and malaria control in low resources areas. This study explores whether accurate Plasmodium species identification-a critical step during the diagnosis protocol in order to choose the appropriate medication-is possible through the information provided by non-trained on-line volunteers. METHODS: 88 volunteers have performed a series of questionnaires over 110 images to differentiate species (Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi) and parasite staging from thin blood smear images digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Visual cues evaluated in the surveys include texture and colour, parasite shape and red blood size. RESULTS: On-line volunteers are able to discriminate Plasmodium species (P. falciparum, P. malariae, P. vivax, P. ovale, P. knowlesi) and stages in thin-blood smears according to visual cues observed on digitalized images of parasitized red blood cells. Friendly textual descriptions of the visual cues and specialized malaria terminology is key for volunteers learning and efficiency. CONCLUSIONS: On-line volunteers with short-training are able to differentiate malaria parasite species and parasite stages from digitalized thin smears based on simple visual cues (shape, size, texture and colour). While the accuracy of a single on-line expert is far from perfect, a single parasite classification obtained by combining the opinions of multiple on-line volunteers over the same smear, could improve accuracy and reliability of Plasmodium species identification in remote malaria diagnosis.