The impact of malaria-protective red blood cell polymorphisms on parasite biomass in children with severe Plasmodium falciparum malaria.

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4.

Tocilizumab for treatment of SARS-CoV-2 infection at home: A case report.

SARS-CoV-2 virtual wards have successfully developed to monitor and escalate patients to hospital throughout the pandemic. Here we describe the case of an 84 year old man who received his complete care for severe SARS-CoV-2 pneumonitis at home, including the administration of oxygen, dexamethasone and tocilizumab.

Forces acting on codon bias in malaria parasites.

Malaria parasite genomes have a range of codon biases, with Plasmodium falciparum one of the most AT-biased genomes known. We examined the make up of synonymous coding sites and stop codons in the core genomes of representative malaria parasites, showing first that local DNA context influences codon bias similarly across P. falciparum, P. vivax and P. berghei, with suppression of CpG dinucleotides and enhancement of CpC dinucleotides, both within and aross codons. Intense asexual phase gene expression in P. falciparum and P. berghei is associated with increased A3:G3 bias but reduced T3:C3 bias at 2-fold sites, consistent with adaptation of codons to tRNA pools and avoidance of wobble tRNA interactions that potentially slow down translation. In highly expressed genes, the A3:G3 ratio can exceed 30-fold while the T3:C3 ratio can be less than 1, according to the encoded amino acid and subsequent base. Lysine codons (AAA/G) show distinctive behaviour with substantially reduced A3:G3 bias in highly expressed genes, perhaps because of selection against frameshifting when the AAA codon is followed by another adenine. Intense expression is also associated with a strong bias towards TAA stop codons (found in 94% and 89% of highly expressed P. falciparum and P. berghei genes respectively) and a proportional rise in the TAAA stop 'tetranucleotide'. The presence of these expression-linked effects in the relatively AT-rich malaria parasite species adds weight to the suggestion that AT-richness in the Plasmodium genus might be a fitness adaptation. Potential explanations for the relative lack of codon bias in P. vivax include the distinct features of its lifecycle and its effective population size over evolutionary time.

The ReSurge Global Training Program: A Model for Surgical Training and Capacity Building in Global Reconstructive Surgery.

BACKGROUND: A paradigm shift is underway in the world of humanitarian global surgery to address the large unmet need for reconstructive surgical services in low- and middle-income countries (LMICs). Here, we discuss the ReSurge Global Training Program (RGTP), a model for surgical training and capacity building in reconstructive surgery in the developing world. The program includes an online reconstructive surgery curriculum, visiting educator trips, expert reconstructive surgeon involvement, trainee competency tracking system, and identification of local outreach partners to provide safe reconstructive surgery to the neediest of patients in the developing world. METHODS: A retrospective review of the components of the RGTP from July 2014 through June 2017 was performed. Trainee milestones scores were analyzed to observe trends toward competency in specific plastic surgery skill sets. RESULTS: There were a total of 38 visiting educator trips during the study period. The trips took place in 10 LMICs. A total of 149 trainees were evaluated in the context of the visiting educator trips with 377 distinct submodule evaluations. Four trainees had more than 10 submodule evaluations over 2 or more visiting educator trips. There was notable improvement in milestones ratings over time among the trainees in this program. CONCLUSIONS: The RGTP is a model of reconstructive surgical training and capacity building in LMICs. Trainees develop important skill sets in reconstructive surgery as a result of their involvement in the program. This comprehensive training approach addresses the disparity in access to care in the developing world by providing short- and long-term solutions to unmet reconstructive needs.

Post-treatment haemolysis in African children with hyperparasitaemic falciparum malaria; a randomized comparison of artesunate and quinine.

BACKGROUND: Parenteral artesunate is the treatment of choice for severe malaria. Recently, haemolytic anaemia occurring 1 to 3 weeks after artesunate treatment of falciparum malaria has been reported in returning travellers in temperate countries. METHODS: To assess these potential safety concerns in African children, in whom most deaths from malaria occur, an open-labelled, randomized controlled trial was conducted in Kinshasa, Democratic Republic of Congo. 217 children aged between 6 months and 14 years with acute uncomplicated falciparum malaria and parasite densities over 100,000/µL were randomly allocated to intravenous artesunate or quinine, hospitalized for 3 days and then followed for 42 days. RESULTS: The immediate reduction in haemoglobin was less with artesunate than with quinine: median (IQR) fall at 72 h 1.4 g/dL (0.90-1.95) vs. 1.7 g/dL (1.10-2.40) (p = 0.009). This was explained by greater pitting then recirculation of once infected erythrocytes. Only 5% of patients (in both groups) had a ≥ 10% reduction in haemoglobin after day 7 (p = 0.1). One artesunate treated patient with suspected concomitant sepsis had a protracted clinical course and required a blood transfusion on day 14. CONCLUSIONS: Clinically significant delayed haemolysis following parenteral artesunate is uncommon in African children hospitalised with acute falciparum malaria and high parasitaemias. TRIAL REGISTRATION: ClinicalTrials.gov ; Identifier: NCT02092766 (18/03/2014).

Formaldehyde as a trigger for protein aggregation and potential target for mitigation of age-related, progressive cognitive impairment.

Recently, formaldehyde (FA), existing in a number of different cells including neural cells, was found to affect age-related cognitive impairment. Oral administration of methanol (the metabolic precursor of FA) triggers formation of senile plaques (SPs) and Tau hyperphosphorylation in the brains of monkeys with memory decline. Intraperitoneal injection of FA leads to hyperphosphorylation of Tau in wild-type mouse brains and N2a cells through activation of glycogen synthase kinase-3ß (GSK-3ß). Furthermore, formaldehyde at low concentrations can directly induce Tau aggregation and amyloid ß (Aß) peptide deposits in vitro. Formaldehyde-induced Tau aggregation is implicated in cytotoxicity and neural cell apoptosis. Clarifying how FA triggers Aß deposits and Tau hyperphosphorlyation will not only improve our understanding of the molecular and cellular mechanisms of age-related cognitive impairment but will also contribute to the ongoing investigation of alternate targets for new drugs. Here, we review the role of FA, particularly that of endogenous origin, in protein aggregation and as a potential drug intervention in the development of agerelated cognitive impairment.

Dietary methanol and autism.

The authors sought to establish whether maternal dietary methanol during pregnancy was a factor in the etiology of autism spectrum disorders. A seven item questionnaire was given to women who had given birth to at least one child after 1984. The subjects were solicited from a large primary care practice and several internet sites and separated into two groups - mothers who had given birth to a child with autism and those who had not. Average weekly methanol consumption was calculated based on questionnaire responses. 550 questionnaires were completed by women who gave birth to a non-autistic child. On average these women consumed 66.71mg. of methanol weekly. 161 questionnaires were completed by women who had given birth to an autistic child. The average estimated weekly methanol consumption for this group was 142.31mg. Based on the results of the Wilcoxon rank sum-test, we see a significant difference between the reported methanol consumption rates of the two groups. This study suggests that women who have given birth to an autistic child are likely to have had higher intake of dietary sources of methanol than women who have not. Further investigation of a possible link of dietary methanol to autism is clearly warranted.

Field-based flow cytometry for ex vivo characterization of Plasmodium vivax and P. falciparum antimalarial sensitivity.

Ex vivo antimalarial sensitivity testing in human malaria parasites has largely depended on microscopic determination of schizont maturation. While this microscopic method is sensitive, it suffers from poor precision and is laborious. The recent development of portable, low-cost cytometers has allowed us to develop and validate a simple, field-optimized protocol using SYBR green and dihydroethidium for the accurate and objective determination of antimalarial drug sensitivity in freshly isolated Plasmodium vivax and Plasmodium falciparum.

Methanol: a chemical Trojan horse as the root of the inscrutable U.

Until 200 years ago, methanol was an extremely rare component of the human diet and is still rarely consumed in contemporary hunter and gatherer cultures. With the invention of canning in the 1800s, canned and bottled fruits and vegetables, whose methanol content greatly exceeds that of their fresh counterparts, became far more prevalent. The recent dietary introduction of aspartame, an artificial sweetener 11% methanol by weight, has also greatly increased methanol consumption. Moreover, methanol is a major component of cigarette smoke, known to be a causative agent of many diseases of civilization (DOC). Conversion to formaldehyde in organs other than the liver is the principal means by which methanol may cause disease. The known sites of class I alcohol dehydrogenase (ADH I), the only human enzyme capable of metabolizing methanol to formaldehyde, correspond to the sites of origin for many DOC. Variability in sensitivity to exogenous methanol consumption may be accounted for in part by the presence of aldehyde dehydrogenase sufficient to reduce the toxic effect of formaldehyde production in tissue through its conversion to the much less toxic formic acid. The consumption of small amounts of ethanol, which acts as a competitive inhibitor of methanol's conversion to formaldehyde by ADH I, may afford some individuals protection from DOC.

Antimalarial drugs: recent advances in molecular determinants of resistance and their clinical significance.

Molecular determinants of antimalarial drug resistance are useful and informative tools that complement phenotypic assays for drug resistance. They also guide the design of strategies to circumvent such resistance once it has reached levels of clinical significance. Established resistance to arylaminoalcohols such as mefloquine and lumefantrine in SE Asia is mediated primarily by gene amplification of the P. falciparum drug transporter, pfmdr1. Single nucleotide polymorphisms in pfmdr1, whether assessed in field isolates or transfection experiments, are associated with changes in IC(50) values (to arylaminoalcohols and chloroquine), but not of such magnitude as to influence clinical treatment outcomes. Recently described emerging in vitro resistance to artemisinins in certain areas correlates with mutations in the SERCA-like sequence PfATP6 and supports PfATP6 as a key target for artemisinins.

Artemisinins.

Artemisinins were discovered to be highly effective antimalarial drugs shortly after the isolation of the parent artemisinin in 1971 in China. These compounds combine potent, rapid antimalarial activity with a wide therapeutic index and an absence of clinically important resistance. Artemisinin containing regimens meet the urgent need to find effective treatments for multidrug resistant malaria and have recently been advocated for widespread deployment. Comparative trials of artesunate and quinine for severe malaria are in progress to see if the persistently high mortality of this condition can be reduced.

Why is the Plasmodium falciparum hexose transporter a promising new drug target?

Chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of Plasmodium falciparum, the causative agent of severe malaria, are wholly dependent upon host glucose for energy. A facilitative hexose transporter (PfHT), encoded by a single-copy gene, mediates glucose uptake and is therefore an attractive potential target. The authors first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. They then used this expression system to compare the interaction of substrates with PfHT and mammalian Gluts (hexose transporters) and identified important differences between host and parasite transporters. Certain Omethyl derivatives of glucose proved to be particularly useful discriminators between mammalian transporters and PfHT. The authors exploited this selectivity and synthesised an O-3 hexose derivative that potently inhibits PfHT expressed in oocytes. This O-3 derivative (compound 3361) also kills cultured P. falciparum with comparable potency. Compound 3361 acts with reasonable specificity against PfHT orthologues encoded by other parasites such as Plasmodium vivax, Plasmodium yoelii and Plasmodium knowlesi. Multiplication of Plasmodium berghei in a mouse model is also significantly impeded by this compound. These findings validate PfHT as a novel target.

Occludin expression in microvessels of neoplastic and non-neoplastic human brain.

The tight junction protein occludin 'glues' normal, adjacent brain microvessel endothelial cells together. Malignant brain tumours cause cerebral oedema because they have leaky endothelial tight junctions, which allow plasma fluid to enter the brain from the microvessel lumen. In order to identify molecular abnormalities in tumour endothelial tight junctions, we investigated occludin expression in microvessels from adult human non-neoplastic brain tissue using immunohistochemistry and immunoblotting. The proportions of microvessels immunolabelling for occludin were >2/3 in 5/5 non-neoplastic brain tissue samples, >1/3 in 5/5 low grade (Daumas-Duport I or II) astrocytomas and <1/3 in 5/5 high grade (III or IV) astrocytomas and 6/6 metastatic adenocarcinomas. Six non-neoplastic brain tissue immunoblots gave a 55-kDa occludin band, three low-grade astrocytomas gave 55-kDa and 60-kDa bands, 13 high-grade astrocytomas gave 60-kDa or no band and four adenocarcinomas did not give an occludin band. Expression of 55-kDa occludin inversely correlated with the presence of contrast enhancement on computed tomograms (P < 0.001). Electron microscopy showed open endothelial tight junctions in 0/2 non-neoplastic human brain specimens and 2/2 high-grade astrocytomas. We suggest that loss of 55-kDa occludin expression in human brain tumours may contribute to endothelial tight junction opening. Characterizing the molecular pathology of brain endothelial tight junctions may facilitate the design of novel drugs against cerebral oedema.

Transport proteins of Plasmodium falciparum: defining the limits of metabolism.

In this review we give an account of transport processes occurring at the membrane interface that separates the asexual stage of Plasmodium falciparum from its host, the infected erythrocyte, and also describe proteins whose activities may be important at this location. We explain the potential clinical value of such studies in the light of the current spread of parasite resistance to conventional antimalarial strategies. We discuss the uptake of substrates critical to the survival of the intracellular malaria parasite, and also the parasite's homeostatic and disposal mechanisms. The use of the Xenopus laevis expression system in the characterisation of a hexose transporter ("PfHT1") and a Ca(2+) ATPase ("PfATP4") of the parasite plasma membrane are described in detail.

Characterization of P-type ATPase 3 in Plasmodium falciparum.

We report the nucleotide sequence, derived amino acid sequence and expression profile of P-type ATPase 3 (PfATPase3) from Plasmodium falciparum. An open reading frame of 7362 nucleotides, interrupted by a single intron of 168 nt, encoded a protein product of 2394 amino acids with a predicted MW of 282791 Da. Hydropathy analysis of PfATPase3 revealed six amino-terminal and six carboxyl-terminal membrane spanning regions (M1-12) flanking a large hydrophilic domain with a smaller hydrophilic loop between M4 and M5. Based on a phylogenetic comparison of conserved domains present in P-type ATPases from other organisms, PfATPase3 resembled a Type-V ATPase for which the transport affinity is unknown. The PfATPase3 topology was interrupted by four regions, termed 'inserts', unique to malarial P-type ATPases, which were high in asparagine residues and charged amino acids (inserts I1-I4). Inserts I1 and I3 also contained repeated amino acid motifs. The number and composition of repeated amino acid motifs in insert I3 were variable in seven P. falciparum strains tested. PfATPase3 was 80.2% similar to the non-insert portions of P. yoelii ATPase3, although their inserts differed in length and composition. PfATPase3 mRNA was most abundant relative to beta-tubulin during the latter half of the erythrocytic cycle and was also present in gametocytes. Using affinity-purified antibody to a 14 amino acid PfATPase3 epitope, a 260 kDa protein was detected by Western analysis. Based on immunofluorescence, the PfATPase3 protein was located intracellularly in gametocytes and, to a lesser extent, in late erythrocytic stages.

Bioavailability and preliminary clinical efficacy of intrarectal artesunate in Ghanaian children with moderate malaria.

We report the first detailed pharmacokinetic assessment of intrarectal (i.r.) artesunate (ARS) in African children. Artesunate was given intravenously (i.v.; 2.4 mg/kg of body weight) and i.r. (10 or 20 mg/kg formulated as 50- or 200-mg suppositories [Rectocaps]) in a crossover study design to 34 Ghanaian children with moderate falciparum malaria. The median relative bioavailability of dihydroartemisinin (DHA), the active antimalarial metabolite of ARS, was higher in the low-dose i.r. group (10 mg/kg) than in the high-dose i.r. group (20 mg/kg) (58 versus 23%; P = 0.018). There was wide interpatient variation in the area under the concentration-time curve after i.r. ARS administration (up to 9-fold in the high-dose group and 20-fold in the low-dose group). i.r. administered ARS was more rapidly absorbed in the low-dose group than the high-dose group (median [range] absorption half-lives, 0.7 h [0.3 to 1.24 h] versus 1.1 h [0.6 to 2.7 h] [P = 0.023]. i.r. administered ARS was eliminated with a median (range) half-life of 0.8 h (0.4 to 2.7 h) (low-dose group and 0.9 h (0.1 to 2.5 h) (high-dose group) (P = 1). The fractional clearances of DHA were 3.9, 2.6, and 1.5 liters/kg/h for the 20-mg/kg, 10-mg/kg and i.v. groups, respectively (P = 0.001 and P = 0.06 for the high-and low-dose i.r. groups compared with the i.v. groups, respectively). The median volumes of distribution for DHA were 1.5 liters kg (20 mg/kg, i.r. group), 1.8 liters/kg (10 mg/kg, i.r. group), and 0.6 liters/kg (i.v. group) (P < 0.05 for both i.r. groups compared with the i.v. group). Parasite clearance kinetics were comparable in all treatment groups. i.r. administered ARS may be a useful alternative to parenterally administered ARS in the management of moderate childhood malaria and should be studied further.

Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms.

We have obtained a full-length P type ATPase sequence (PfATP4) encoded by Plasmodium falciparum and expressed PfATP4 in Xenopus laevis oocytes to study its function. Comparison of the hitherto incomplete open reading frame with other Ca(2+)-ATPase sequences reveals that PfATP4 differs significantly from previously defined categories. The Ca(2+)-dependent ATPase activity of PfATP4 is stimulated by a much broader range of [Ca(2+)](free) (3.2-320 micrometer) than are an avian SERCA1 pump or rabbit SERCA 1a (maximal activity < 10 micrometer). The activity of PfATP4 is resistant to inhibition by ouabain (200 micrometer) or thapsigargin (0.8 micrometer) but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 microM). We used a quantitative polymerase chain reaction to assay expression of mRNA encoding PfATP4 relative to that for beta-tubulin in synchronized asexual stages and found variable expression throughout the life cycle with a maximal 5-fold increase in meronts compared with ring stages. This analysis suggests that PfATP4 defines a novel subclass of Ca(2+)-ATPases unique to apicomplexan organisms and therefore offers potential as a drug target.

Hexose transport in asexual stages of Plasmodium falciparum and kinetoplastidae.

The hexose sugar, glucose, is a vital energy source for most organisms and an essential nutrient for asexual stages of Plasmodium falciparum. Kinetoplastid organisms (e.g. Trypanosoma and Leishmania spp) also require glucose at certain critical stages of their life cycles. Although phylogenetically unrelated, these organisms share many common challenges during the mammalian stages of a parasitic life cycle, and possess hexose uptake mechanisms that are amenable to study using similar methods. Defining hexose permeation pathways into parasites might expose an Achilles' heel at which both antidisease and antiparasite measures can be aimed. Understanding the mode of entry of glucose also presents a good general model for substrate acquisition in multicompartment systems. In this review, Sanjeev Krishna and colleagues summarize current understanding of hexose transport processes in P. falciparum and provide a comparison with data obtained from kinetoplastids.

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, K(m) approximately 1.0 mM) also transports fructose (K(m) approximately 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.

Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.

Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter of P. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (Km = 0.48 mM) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition.