Biology subjects

Baum, J.

Publications and source records attributed to Baum, J..

43 records · Page 3Linked to original sources

bioRxiv2019

Quantitative and rapid Plasmodium falciparum malaria diagnosis and artemisinin-resistance detection using a CMOS Lab-on-Chip platform

Early and accurate diagnosis of malaria and drug-resistance is essential to effective disease management. Available rapid malaria diagnostic tests present limitations in analytical sensitivity, drug-resistant testing and/or quantification. Conversely, diagnostic methods based on nucleic acid amplification stepped forwards owing to their high sensitivity, specificity and robustness. Nevertheless, these methods commonly rely on optical measurements and complex instrumentation which limit their applicability in resource-poor, point-of-care settings. This paper reports the specific, quantitative and fully-electronic detection of Plas-modium falciparum, the predominant malaria-causing parasite worldwide, using a Lab-on-Chip platform developed in-house. Furthermore, we demonstrate on-chip detection of C580Y, the most prevalent single-nucleotide polymorphism associated to artemisinin-resistant malaria. Real-time non-optical DNA sensing is facilitated using Ion-Sensitive Field-Effect Transistors, fabricated in unmodified complementary metal-oxide-semiconductor technology, coupled with loop-mediated isothermal amplification. This work holds significant potential for the development of a fully portable and quantitative malaria diagnostic that can be used as a rapid point-of-care test.

bioengineering↗

bioRxiv2019

The effects of dyslipidaemia and cholesterol modulation on erythrocyte susceptibility to malaria parasite infection

Malaria disease commences when blood-stage parasites, called merozoites, invade human red blood cells (RBCs). Whilst the process of invasion is traditionally seen as being entirely merozoite-driven, emerging data suggests RBC biophysical properties markedly influence invasion. Cholesterol is a major determinant of cell membrane biophysical properties. We set out to assess whether cholesterol content in the RBC membrane affects susceptibility to merozoite invasion. Here we demonstrate that RBC bending modulus (a measure of deformability) is markedly affected by artificial modulation of cholesterol content and negatively correlated with merozoite invasion efficiency. Contextualising this observation, we tested a mouse model of hypercholesterolemia and human clinical samples from patients with a range of serum cholesterol concentrations for parasite susceptibility. Hypercholesterolaemia in both human and murine subjects had little effect merozoite invasion efficiency. Furthermore, on testing, RBC cholesterol content in both murine and human hypercholesterolaemia settings was found to be unchanged from normal controls. Serum cholesterol is, therefore, unlikely to impact on RBC susceptibility to merozoite entry. Our work, however, suggests that native polymorphisms that affect RBC membrane lipid composition would be expected to affect parasite entry. This supports investigation of RBC biophysical properties in endemic settings, which may yet identify naturally protective lipid-related polymorphisms.

microbiology↗

bioRxiv2019

Increased Dynamics of α-Synuclein Fibrils by β-Synuclein Leads to Reduced Seeding and Cytotoxicity

Alpha-synuclein (S) fibrils are toxic to cells and contribute to the pathogenesis and progression of Parkinsons disease and other synucleinopathies. {beta}-Synuclein ({beta}S), which co-localizes with S, has been shown to provide a neuroprotective effect, but the molecular mechanism by which this occurs remains elusive. Here we show that S fibrils formed in the presence of {beta}S are less cytotoxic, exhibit reduced cell seeding capacity and are more resistant to fibril shedding compared to S fibrils alone. Using solid-state NMR, we found that the overall structure of the core of S fibrils when co-incubated with {beta}S is minimally perturbed, however, the dynamics of Lys and Thr residues, located primarily in the imperfect KTKEGV repeats of the S N-terminus, are increased. Our results suggest that amyloid fibril dynamics may play a key role in modulating toxicity and seeding. Thus, enhancing the dynamics of amyloid fibrils may be a strategy for future therapeutic targeting of neurodegenerative diseases.

biophysics↗

bioRxiv2018

Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

Invasion of and intracellular development within the red blood cell by malaria parasites requires interaction with a multitude of host proteins expressed at the surface of or inside the cell. Perhaps the biggest obstacle to dissection of specific functions of host proteins during invasion is the inability to manipulate protein expression in the genetically intractable anucleate red blood cell. Whilst genetic manipulation and subsequent in vitro differentiation of nucleated erythroid precursors have facilitated progress in this area, such approaches are limited by the finite proliferative capacity of primary hematopoietic stem cells, and a failure of erythroleukemic cell lines to enucleate, respectively. Here, we report that reticulocytes derived through in vitro differentiation of an enucleation competent immortalized erythroblast cell line (BEL-A) support both successful invasion and growth by Plasmodium falciparum. Using CRISPR-mediated gene knockout and lentiviral expression of open reading frames, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and, for the first time, demonstrate that this can be rescued by re-expression of the receptor or of a mutant thereof. Specifically, using sustainable edited clones derived from this line, we exclude a functional role for the cytoplasmic domain of basigin during invasion, and challenge the reported requirement of its associated receptor cyclophilin B. These data establish the use of reticulocytes derived from immortalized erythroblasts as a crucial model system to explore specific hypotheses regarding host receptor requirements and involvement in P. falciparum invasion.

cell biology↗

bioRxiv2018

A high-throughput in vitro translation screen towards discovery of novel antimalarial protein translation inhibitors

Drugs that target protein synthesis are well-validated for use as antimicrobials, yet specific high throughput (HTP) methods to screen for those targeting malaria are lacking. Here, we have developed a cell free in vitro translation (IVT) assay for the human malaria parasite, Plasmodium falciparum, which reconstitutes the native parasite protein translation machinery. Combining clarified IVT lysate with a click beetle luciferase reporter gene fused to untranslated regions of Pf histidine-rich proteins (hrp)-2 and 3, the HTP IVT assay accurately reports protein translation in a 384-well plate format using a standard spectrofluorometer. We validate the assay as effective in detecting compounds targeting the ribosome, ribosome co-factors (elongation factor 2) and cytosolic tRNA synthetases as well as its ability to find translation inhibitors in a blind screen using a high-density assay format amenable for high throughput. This demonstrates an ability to reconstitute the breadth of the parasite eukaryotic protein translation machinery in vitro and use it as a powerful platform for antimalarial drug discovery.

microbiology↗

bioRxiv2017

Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

A critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum, has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro. In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei. Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro. Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

microbiology↗

bioRxiv2017

Binding Of A Newly Identified Essential Light Chain To Expressed Plasmodium falciparum Class XIV Myosin Enhances Actin Motility

Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional \"essential\" light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 m/sec, more than twice that of PfMyoA-MTIP (~1.7 m/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.\n\nSignificanceMotility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, relies on a divergent actomyosin system powered by the class XIV myosin, PfMyoA. We show that functional PfMyoA can be expressed in Sf9 cells if a Plasmodium spp. myosin chaperone is co-expressed. We identified an \"essential\" light chain (PfELC) that binds to PfMyoA in parasites. In vitro expression of PfMyoA heavy chain with PfELC and the known light chain MTIP produced the fastest speeds of actin movement (~3.8 m/sec). Duty ratio estimates suggest that ~6 PfMyoA motors can move actin at maximal speed, a feature that may facilitate interaction with short, dynamic Plasmodium actin filaments. Our findings enable drug screening for myosin-based inhibitors of Plasmodium cellular invasion.

biochemistry↗