Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum.

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.

Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation.

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

High-Content Phenotypic Screen of a Focused TCAMS Drug Library Identifies Novel Disruptors of the Malaria Parasite Calcium Dynamics.

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.

Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics.

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

A Pilot Study Using Intratympanic Methylprednisolone for Treatment of Persistent Posterior Canal Benign Paroxysmal Positional Vertigo.

OBJECTIVE: To assess the effect of intratympanic methylprednisolone (ITMP) in posterior canal benign paroxysmal positional vertigo (BPPV) that fails treatment involving repositioning maneuver in a case series. MATERIALS AND METHODS: Nine patients with persistent posterior canal BPPV after 6 or more repositioning maneuvers were treated by ITMP (two weekly doses of 0.3-0.4 mL at 40 mg/mL) before repeating the repositioning procedures. RESULTS: Following ITMP treatment, 7 out of 9 patients were relieved of their symptoms and did not exhibit positional nystagmus after 1 or 2 repositioning maneuvers. The number of positional maneuvers performed before and after ITMP treatment in these 7 patients showed a statistically significant (p=0.016) reduction in the amount of repositioning treatments required. None of the 7 respondent patients showed any relapses during the follow-up period (follow-up range: 11-95 months). CONCLUSION: Administering ITMP before resuming repositioning procedures can be a useful treatment for persistent BPPV of the posterior canal.

A broad analysis of resistance development in the malaria parasite.

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.

Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay.

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compounds that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compound libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. METHODS: Parasite killing kinetics were determined by first culturing unlabelled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes pre-labelled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labelled erythrocytes could then be detected by two-colour flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labelled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be determined. RESULTS: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs versus those acting more slowly. The assay was validated against ten standard anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaption to medium-high throughput screening, was validated and applied against a set of 20 compounds retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. CONCLUSION: The quantification of new infections to determine parasite viability offers important advantages over existing methods, and is amenable to medium-high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.

Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence "transmission blocking" assay.

BACKGROUND: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. METHODS AND FINDINGS: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. CONCLUSIONS: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.

Recurrence of benign paroxysmal positional vertigo.

OBJECTIVE: To determine the recurrence rate of benign positional paroxysmal vertigo (BPPV) and the factors associated to such recurrences. STUDY DESIGN: Prospective study. METHOD: Sixty-nine consecutive patients treated for first episode of BPPV. STUDY PERIOD: 63 months. Mean follow-up: 47 months. RESULTS: The recurrence rate was 27%. Fifty percent of recurrences occurred in the first 6 months. Nineteen patients had 1 or more recurrence of BPPV; 10 had 1 recurrence, 7 patients had 2, and 2 patients had 3 recurrences. There was no significant difference in the recurrence rate according to sex, age, side, cause of BPPV, or instability after successful treatment. Multi-canal BPPV (log-rank, p = 0.024) and anterior canal BPPV (log-rank, p = 0.029) showed a significantly greater tendency to recur and to do so earlier. There was a significant difference in time to recurrence related to the number of maneuvers used to resolve the initial BPPV episode (log-rank, p = 0.023). Except for cases of BPPV secondary to labyrinthitis or neurolabyrinthitis, at least 70% of the recurrences affected a different side and/or different canal than the primary BPPV. CONCLUSION: The recurrence rate of BPPV is 27%, and relapse largely occurs in the first 6 months. When BPPV recurrence is suspected, every canal on both sides must be investigated because it is the BPPV syndrome that recurs, rather than BPPV affecting a particular side or canal. Complex cases of BPPV have a greater risk of recurrence.

In vivo UVB-photoprotective activity of extracts from commercial marine macroalgae.

The photoprotective potential against UVB radiation of extracts obtained from 21 commercial macroalgae from the Phyla Ochrophyta and Rhodophyta, was evaluated in vivo, using the zebrafish embryo as a whole model organism. Our results showed that the phenolic extracts from Macrocystis pyrifera and Porphyra columbina exhibited the highest photoprotective activity, close to complete photoprotection (100%), similar to that obtained for the carrageenophytes Sarcothalia radula and Gigartina skottsbergii. Under the assayed conditions, the extracts were safe and non-toxic to the embryos at a concentration of 0.04 mg/ml PGE.

Efficacy of routine bilateral neck dissection in the management of the N0 neck in T1-T2 unilateral supraglottic cancer.

BACKGROUND: The treatment of the clinically negative (N0) neck in supraglottic laryngeal cancer continues to be an area of controversy. The aim of this study was to analyze the long-term efficacy of routine bilateral neck dissection compared with ipsilateral neck dissection in T1-T2 N0 lateral supraglottic carcinomas. METHODS: A retrospective review of 108 patients who underwent surgery for T1-T2 supraglottic squamous cell carcinoma was performed. Forty-eight had undergone ipsilateral functional neck dissection, and 60 had undergone bilateral functional neck dissections. None of these patients received adjuvant radiotherapy. RESULTS: No significant differences (p = .78) in regional recurrence were observed between the patients treated with bilateral neck dissection (13%) and those treated with ipsilateral neck dissection (17%). The 5-year survival rates were 73% and 80% for the patients who received a bilateral and ipsilateral neck dissection, respectively (p = .51). CONCLUSIONS: This study suggests that routine bilateral neck dissection may not be necessary in the surgical treatment of all supraglottic cancers.

Avaliaçäo antropométrica e perfil plasmático de ácidos graxos de recém-nascidos pré-termo submetidos a duas dietas isocalóricas e com diferentes composiçoes lipídicas / Anthropometric evaluation and plasm fatty acids profile of preterm newborn infants subjected 2 isocaloric diets of different lipid composition

Este trabalho analisou o perfil de ácidos graxos plamáticos eo crescimento somático de 20 recém-nascidos pré-termos, com idade gestacional entre 30 a 35 semanas, admitidos no Berçário do Hospital das Clínicas da Faculdade de Medicina de Ribeiräo Preto da Universidade de Säo Paulo, submetidos, por um período de aproximadamente 20 dias, a duas dietas isocalóricas e com diferentes composiçöes liídicas. Um grupo de 10 crianças GRUPO L.D.S. usou uma dieta contendo 0,5% das calorias totais (54,5 mg/100 cal) como ácido linoléico (C18:2) e relaçäo PS=0,053. Os outros 10 recém-nascidos GRUPO L.D.O. foram colocados numa dieta contendo 10,5% das calorias totais (1171mg/100 kcal) como ácido linoléico (C18:2) e a relaçäo P/S = 0,508. Os grupos no período imediato antes da instituiçäo das dietas em estudo apresentaram valores plasmáticos dos ácidos linoléico (C18:2) e aracdônico (20:4) semelhantes entre si e superiores aos limites mínimos normais estabelecidos por WIESE etalii (1958) e HANSEN ET ALII (1963), refletindo reservas corporais e dietas semelhantes previamente ao estudo. Após o uso da dieta no GRUPO L.D.S. houve uma queda significante (p < 0,05) dos níveis plasmáticos ácidos C16:0 (palmítico), C16:1 (palmitoléico) e C18:1 (oléico) se elevaram significantemente (p < 0,05). As alteraçöes encontradas foram semelhantes as relatadas em diversos trabalhos da literatura e poderiam ser considerados como evidências bioquímicas de deficiência de ácidos graxos essenciais. No GRUPO L.D.O. após o uso da dieta verificamos uma queda nos níveis plasmáticos do ácido aracdônico (C20:4 a níveis inferiores aos mínimos normais estabelecidos na literatura em 90% dos recém-nascidos pré-termos, mas a queda foi menor do que a verificada no GRUPO L.D.S. (p < 0,,5). O ácido linoléico (C18:2) apresentou elevaçäo no seu nível plasmático em 50% dos pré-termos estudados e nos outros 50% os valores foram semelhantes ou ligeiramente inferiores ao período prédieta, mas todos apresentaram níveis superiores aos limites mínimos normais e aos do GRUPO L.D.S. (p , 0,05). Os niveis plasmticos elevados de C18:2 no GRUPO L.D.O. após o uso da dieta com elevado conteúdo de ácido linoléico concordam com a melhor absorçäo de gorduras verificada neste grupo em relaçäo ao GRUPO L.D.S. (p < 0,05)...

Gastric emptying in children: IV. Studies on kwashiorkor and on marasmic kwashiorkor

O esvaziamento gástrico de uma refeiçäo líquida foi estudado em 22 crianças com desnutriçäo protéico-calórica do III- grau que foram divididas em dois grupos, de acordo com o tipo clínico apresentado: kwashiorkor (n = 11) e kwashiorkor-marasmático (n = 11). O esvaziamento gástrico foi avaliado pela técnica da dupla amostragem, em diferentes instantes após a instilaçäo intragástrica de glicose a 5%, no volume de 20 ml/kg de peso corporal. O grupo controle foi constituído por sete crianças sadias. Nas crianças desnutridas, o estudo do esvaziamento gástrico foi realizado nas primeiras 72 horas após a admissäo e 30 dias após, quando o estado nutricional já estava em franca recuperaçäo. Os integrantes do grupo controle foram submetidos a um único teste. Os volumes restantes no estômago das crianças com kwashiorkor nos diferentes instantes do primeiro estudo näo diferiram significativamente dos obtidos quando a recuperaçäo nutricional já havia se iniciado. Nas crianças com kwashiorkor-marasmático recém-admitidas, os volumes restantes no estômago 30 minutos após a administraçäo da refeiçäo, foram significativamente maiores que nos controles, mas a diferença desapareceu após o início da recuperaçäo nutricional. Estes resultados indicam que as crianças com kwashiorkor näo apresentam anormalidades do esvaziamento gástrico de líquidos, enquanto que nas crianças com kwashiorkor-marasmático o esvaziamento é retardado, mas o distúrbio é reversível com recuperaçäo do estado nutricional

Marcadores sorológicos da hepaite viral B e alfa 1 antitripsina em indios da tribo Mekranhotire / Serological markers for hepatitis B and alpha 1-antitrypsin in Indians of the Mekranhotire tribe

Os autores estudaram marcadores sorológicos do vírus B da hepatite e alfa1 antitripsina em 80 índios da tribo Mekranhotire, grupo Jê, localizados no sul do Pará, à cabeceira do rio Iriri. Foi encontrada positividade de 8,75% para o AgHBs, 91,2% para o anti-HBc, 11,5% para o anti-HBs e 0,0% para AgHHe entre os 80 índios estudados. O grupo controle mostrou positividade em 1% para o AgHBs. 15% para o anti-HBc, 24,7% para o anti-HBs e 0% para AgHBe. Verificaram diferenças significantes quanto aos marcadores AgHBs e anti-HBc. Näo encontraram diferenças significativas quanto ao anti-HBs e AgHBe. As taxas de alfa1 antitripsina foram menores na populaçäo indígena, quando comparadas às do grupo controle