Search

Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes. Supplementary Information The online version contains supplementary material available at 10.1186/s13073-021-00889-9.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-μL min-1 flow rates and105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 μm) that are created by metal patterned over the entire depth (50 μm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (μL min-1 flow and106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes.

Single cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed base content. Through optimization of a quasi-linear genome amplification method, we achieve better targeting of the parasite genome over contaminants and generate coverage levels that allow detection of relatively small copy number variations on a single cell level. These improvements are important for expanding accessibility of single cell approaches to new organisms and for improving the study of adaptive mechanisms.

Corticosteroids are critical for normal development and for mediating effects of stress during development in all vertebrates. Even though gene knockout studies in mouse and zebrafish have identified a number of developmental roles of corticosteroids and their receptors, the numerous pleiotropic actions of these hormones affecting various aspects of development are understudied. For the most part, neither the endogenous hormone(s) nor their receptor(s) regulating developmental processes during natural development have been determined. Here, we address this issue by elucidating the endogenous regulation of the transcription factor Krüppel-like factor 9 (klf9) across tissues during development by corticosteroid hormones (aldosterone and corticosterone) and their nuclear receptors (type-I and type-II receptors). First, we measured the developmental expression profiles of klf9 and type-I and type-II corticosteroid receptors in key target tissues, brain, lungs, and tail, during larval and metamorphic stages in Xenopus tropicalis. We also studied the corticosteroid regulation of klf9 in these tissues in-vivo using exogenous hormone treatments and receptor antagonists. Klf9 and the corticosteroid receptors were expressed in each tissue and significantly increased in expression reaching a peak at metamorphic climax, except for the type-II receptor in brain and tail whose expression did not change significantly across stages. Both corticosteroid hormones induced klf9 in each tissue, although aldosterone required a five times higher dose than corticosterone to cause a significant induction. The upregulation of klf9 by both corticosteroids was completely blocked by the use of the type-II receptor antagonist RU486 and not the type-I receptor antagonist spironolactone. These results are consistent with previous in-vitro studies and indicate for the first time in-vivo that corticosteroid regulation of klf9 occurs exclusively via corticosterone and type-II receptor interaction across tissues.

Plasmodiumprotozoan parasites undergo rounds of asexual replication inside human erythrocytes, progressing from ring stage, to trophozoites and schizonts, before egress and reinvasion. Given the discovery of ring-specific artemisinin tolerance and quiescence inPlasmodium falciparum, there is great urgency to better understand ring stage biology. However, the lack of an effective enrichment method has left rings and related parasite stages understudied compared to their late stage counterparts, which can be easily isolated due to their paramagnetic properties. Here, a method for separatingall Plasmodiuminfected erythrocytes from uninfected erythrocytes is presented. This approach takes advantage of streptolysin-O (SLO) to preferentially lyse uninfected erythrocytes as previously shown by Jackson,et al.Following lytic treatment, Percoll gradient centrifugation removes lysed cells, leaving an intact cell population enriched in infected erythrocytes. ThisSLO-Percoll (SLOPE) method is effective on stages from the entire erythrocytic cycle, including previously inaccessible forms such as circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. Furthermore, the utility of SLOPE is extended to multiple media formulations used for the propagation of two humanPlasmodiumspecies. The alteration of external cholesterol levels modulates SLOPE effectiveness, demonstrating the role of erythrocyte membrane cholesterol in lytic discrimination. Importantly, enrichment does not impact parasite viability, which establishes the non-toxic nature of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms the impact on treated samples; parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples.ImportanceMalaria is caused by infection with protozoanPlasmodiumparasites and is responsible for over 400,000 deaths annually. The availability of effective antimalarial drugs is critical to the reduction of malaria-related mortality, yet widespread resistance highlights the need for the continued study ofPlasmodiumbiology. The SLOPE method is an accessible, scalable, rapid (30-40min), and non-toxic enrichment method that is broadly effective on many erythrocytic stages. This method is ideal for use upstream of a variety of sensitive analyses, which will increase experimental quality in virtually all areas of asexualPlasmodiumparasite research. Further, because the consumption of cholesterol is a common characteristic of other intracellular parasites (both bacteria and other protozoa), SLOPE holds potential for extension to other relevant pathogens.

Sleep loss increases inflammatory mediators in brain and peripheral tissues, but the mechanisms underlying this association are not fully understood. Male C57BL/6j mice were exposed to paradoxical sleep deprivation (PSD) for 24 h using the modified multiple platform (MMP) technique (platforms over water) or two different controls: home cage or a dry platform cage, which constituted a novel environment. PSD mice exhibited increased IL-1β and TNF-α pro-inflammatory gene expression in brain (hypothalamus, hippocampus, pre-frontal cortex), as well as in peripheral tissues (liver, spleen), when compared with home-cage controls. In addition, among PSD mice, TGFβ1, an anti-inflammatory cytokine, was increased in pre-frontal cortex, liver, and spleen in conjunction with elevated serum corticosterone concentration relative to home-cage controls. However, these differences were nearly abolished when PSD mice were compared with control mice subjected to a dry MMP cage, suggesting that simply exposing mice to a novel environment can induce an acute inflammatory response.