Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

<b>Background</b> Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.<p></p> <b>Methods</b> A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.<p></p> <b>Results</b> The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.<p></p> <b>Conclusion</b> The method allows for the enrichment of red blood cells early after the invasion by P. falciparumparasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.<p></p&gt

Translational regulation in Plasmodium falciparum

Plasmodium falciparum is the causative agent of the most malignant form of human malaria, which remains as one of the most devastating infectious diseases. In face of a continuous international effort to eliminate the disease, the parasite not only has evaded a total obliteration, but has now evolved resistance to many of the available drugs. Next generation rational drug design is in urgent need and the key of such will lie on the successful identification of the parasite’s ‘Achilles heel’. While many existing and outstanding drugs have shown the promises of targeting the parasite translation machinery, the translation dynamics as well as the translational regulatory mechanisms are poorly understood. The studies described in this thesis aim to further our understanding on the translational regulation in P. falciparum, at both the global and gene-specific levels. Pregnancy associated malaria (PAM) is commonly seen with excessive sequestration of infected red blood cells in the placenta, the phenomenon is widely considered as the result of the specific ligand-receptor binding between the parasite derived PfEMP1- VAR2CSA proteins and the CSA proteoglycans. Translation of VAR2CSA protein is repressed by an upstream open reading frame, and a predicted trans factor is required for de-repression of var2csa translation. By using a spontaneously derived mutant that fails to efficiently translation the V AR2CSA proteins, we identified PTEF (Plasmodium translation enhancing factor) as the putative trans acting factor that allows efficient VAR2CSA translation. PTEF binds to the ribosomes and can enhance translation in a E. coli system. Importantly, higher PTEF expression was invariably observed to be associated with PAM in previous studies. Furthermore, PTEF function requires the processing by a calpain protease, blockage of the processing abolishes PTEF function in a reporter assay. Our data strongly suggest PTEF is an important regulator of PAM and raises potential therapeutic opportunity. It has been well described that codon usage bias could have a profound effect on translation efficiency. Codon usage is extremely biased in P. falciparum and cumulated to frequent insertions of asparagine homorepeats in up to one fourth of the proteome. However, the biological effect of this codon usage bias has not been studied. By using rationally recodonized GFP sequences, we showed that the increased use of GU wobble codon could reduce translation efficiency. We also demonstrated that the GU wobble- rich codon context underlying the asparagine homorepeats could impart significant influence on the translational output and transcript stability of the host gene. Despite this, GU wobble codons are overrepresented in the genome. Bioinformatics analyses suggested the high content of GU wobble codon might serve as a global regulatory mechanism. We thus offered new insight on the genome evolution of the parasite. RIFIN is the largest variable surface antigen family in P. falciparum. Its research profile has been much uplifted recently, as report showed that it might have a crucial link with severe malaria. While there is a sufficient interest to investigate the regulatory mechanisms associated with the RIFIN family, functional study of RIFIN is often marred by the lack of robustly verified reagents. By using RNA-sequencing and ultra- dense peptide microarray, we were able to authenticate specific RIFIN antibodies that exhibit some degree of intra-family cross-reactivity but minimal non-specific reactivity with other antigens. The derivation of these reagents will be important for future studies

Essays in capital markets

Thesis (Ph.D.)--Massachusetts Institute of Technology, Sloan School of Management, 2002.Includes bibliographical references (p. 136-141).(cont.) Slow information diffusion can cause return momentum. Institutions are thought to be more informed than individuals, and should eliminate return predictability. However, higher institutional ownership is associated with more momentum. Therefore, institutions either herd on returns or can have information before individuals. I find evidence of the latter. However, the effects are economically small, suggesting that aggregate data obscures differences between institutions. I divide institutions by trading aggressiveness. Aggressive institutions are more responsive to recent returns, and a strategy mimicking their trades generates even better performance. This confirms that some investors are more informed than others, but do not eliminate return predictability.This thesis consists of three chapters, each about a separate aspect of how investors respond to information in equity markets. The first chapter concerns news and stock returns. Using a comprehensive database of headlines about individual companies, I examine monthly returns following public news. I compare them to stocks with similar returns, but no identifiable public news. There is a difference between the two sets. I find strong drift after bad news. Investors seem to react slowly to this information. I also find reversal after extreme price movements unaccompanied by public news. The separate patterns appear even after adjustments for risk exposure and other effects. They are, however, mainly seen in smaller, more illiquid stocks. These findings support some integrated theories of investor over- and underreaction. The second chapter is joint work with Richard Frankel and S. P. Kothari. Models based on psychology can explain momentum and reversal in stock returns, but may be overfitted to data. We examine a typical basis for these models, representativeness, in which individuals predict the future based on how closely past outcomes fit certain categories. We use accounting performance to mimic possible investor-defined categories for firm performance. We test the idea that investors predictably bias their expectations about future operations by using these categories. We find little evidence that the sequence or trend of past accounting performance is related to future returns, and is therefore unlikely to bias investor expectations. The third chapter concerns how informational advantage differs between institutional investors.by Wesley S. Chan.Ph.D

Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications

The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality

Prediction of paraquat exposure and toxicity in clinically ill poisoned patients: a model based approach

Aims Paraquat poisoning is a medical problem in many parts of Asia and the Pacific. The mortality rate is extremely high as there is no effective treatment. We analyzed data collected during an ongoing cohort study on self-poisoning and from a randomized controlled trial assessing the efficacy of immunosuppressive therapy in hospitalized paraquat-intoxicated patients. The aim of this analysis was to characterize the toxicokinetics and toxicodynamics of paraquat in this population. Methods A non-linear mixed effects approach was used to perform a toxicokinetic/toxicodynamic population analysis in a cohort of 78 patients. Results The paraquat plasma concentrations were best fitted by a two compartment toxicokinetic structural model with first order absorption and first order elimination. Changes in renal function were used for the assessment of paraquat toxicodynamics. The estimates of toxicokinetic parameters for the apparent clearance, the apparent volume of distribution and elimination half-life were 1.17 l h−1, 2.4 l kg−1 and 87 h, respectively. Renal function, namely creatinine clearance, was the most significant covariate to explain between patient variability in paraquat clearance.This model suggested that a reduction in paraquat clearance occurred within 24 to 48 h after poison ingestion, and afterwards the clearance was constant over time. The model estimated that a paraquat concentration of 429 μg l−1 caused 50% of maximum renal toxicity. The immunosuppressive therapy tested during this study was associated with only 8% improvement of renal function. Conclusion The developed models may be useful as prognostic tools to predict patient outcome based on patient characteristics on admission and to assess drug effectiveness during antidote drug development

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell divisio

The EPIC-Norfolk Eye Study: rationale, methods and a cross-sectional analysis of visual impairment in a population-based cohort.

To summarise the methods of the European Prospective Investigation of Cancer (EPIC)-Norfolk Eye Study, and to present data on the prevalence of visual impairment and associations with visual impairment in the participants

Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research