This book attempts to achieve a difficult goal: to teach statistics to the novice so as to impart a liking and understanding of statistics. The book is geared toward a medical audience, since most examples are from the medical literature. The structure of the book consists of the following elements in each chapter: a small number of statistical rules of thumb, followed by a nontechnical explanation, a demonstration of how to work through the mechanics of doing the statistical test in question, a summary, and sample problems to be solved by the reader. (The answers, with explanations, are provided in an appendix.) Although a variety of univariate statistics are included, certain topics that are important in medical research are not. For example, there is little or no discussion of multiple regression, life-table techniques, or pooling of studies. These omissions, especially of multiple regression, are unfortunate. The Primer was derived from

Primer of Biostatistics

Highly parallel single-cell transcriptome profiling of Plasmodium falciparum blood stages provides insight into the role AP2-G plays in early sexual development of this eukaryotic pathogen. Mosquito-borne transmission of the malaria parasite requires a sub-population of the asexual form of the parasite to differentiate into sexual stages called gametocytes. This process is controlled by the transcriptional regulator AP2-G, but the mechanism involved is poorly understood. In this first application of single-cell RNA sequencing to a eukaryotic pathogen, Bjorn Kafsack and colleagues determine the transcriptional changes induced by AP2-G, identifying other AP2 transcription factors, histone-modifying enzymes and regulators of nucleosome positioning. These findings form the basis for further work aimed at elucidating the mechanistic underpinnings of gametocytogenesis in this important pathogen. Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes1. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48 hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes2,3. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600)4,5, the master regulator of sexual development, from an epigenetically silenced state during asexual replication6,7. AP2-G expression during this ‘commitment cycle’ prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism2,4. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1–30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling8. Here we use highly parallel, single-cell RNA sequencing9 of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.

/pdf/single-cell-rna-sequencing-reveals-a-signature-of-sexual-2h2stgm13w.pdf

Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites

Plasmodium parasites, the causative agents of malaria, have developed elaborate strategies that they use to survive and thrive within different intracellular environments. During the blood stage of infection, the parasite is a master renovator of its erythrocyte host cell, and the changes in cell morphology and function that are induced by the parasite promote survival and contribute to the pathogenesis of severe malaria. In this Review, we discuss how Plasmodium parasites use the protein trafficking motif Plasmodium export element (PEXEL), protease-mediated polypeptide processing, a novel translocon termed the Plasmodium translocon of exported proteins (PTEX) and exomembranous structures to export hundreds of proteins to discrete subcellular locations in the host erythrocytes, which enables the parasite to gain access to vital nutrients and to evade the immune defence mechanisms of the host.

Plasmodium species: master renovators of their host cells

SUMMARY Bovine babesiosis caused by the tick-transmitted haemoprotozoans Babesia bovis, Babesia bigemina and Babesia divergens commonly results in substantial cattle morbidity and mortality in vast world areas. Although existing live vaccines confer protection, they have considerable disadvantages. Therefore, particularly in countries where large numbers of cattle are at risk, important research is directed towards improved vaccination strategies. Here a comprehensive overview of currently used live vaccines and of the status quo of experimental vaccine trials is presented. In addition, pertinent research fields potentially contributing to the development of novel non-live and/or live vaccines are discussed, including parasite antigens involved in host cell invasion and in pathogen-tick interactions, as well as the protective immunity against infection. The mining of available parasite genomes is continuously enlarging the array of potential vaccine candidates and, additionally, the recent development of a transfection tool for Babesia can significantly contribute to vaccine design. However, the complication and high cost of vaccination trials hinder Babesia vaccine research, and have so far seriously limited the systematic examination of antigen candidates and prevented an in-depth testing of formulations using different immunomodulators and antigen delivery systems.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0031182014000961

Vaccines against bovine babesiosis: where we are now and possible roads ahead.

Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

/pdf/an-aspartyl-protease-defines-a-novel-pathway-for-export-of-1mwymracux.pdf

An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/cmi.12460

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles

The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1β branch of the ves multigene family

Rapid clonal antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1) protein expressed on the infected-erythrocyte surface. Because of the significance of this heterodimeric protein for demonstrated mechanisms of parasite survival and virulence, there is a need to understand how expression of the ves multigene family encoding this protein is controlled. As an initial step toward this goal, we present here initial characterization of the ves promoter driving transcription of VESA1a and -1b subunits. A series of transfection constructs containing various sequence elements from the in vivo locus of active ves transcription (LAT) were used to drive expression of the firefly luciferase gene in a dual luciferase-normalized assay. The results of this approach reveal the presence of two bidirectional promoter activities within the 434-bp intergenic region (IGr), influenced by putative regulatory sequences embedded within the flanking ves1α and ves1β genes. Repressor-like effects on the apposing gene were observed for intron 1 of both ves1α and ves1β. This effect is apparently not dependent upon intronic promoter activity and acts only in cis. The expression of genes within the ves family is likely modulated by local elements embedded within ves coding sequences outside the intergenic promoter region in concert with chromatin modifications. These results provide a framework to help us begin to understand gene regulation during antigenic variation in B. bovis.

Characterization of the Unusual Bidirectional ves Promoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

/pdf/knockout-of-babesia-bovis-rad51-ortholog-and-its-3ysksmqmms.pdf

Yu-Ping Xiao

Papers

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles

The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1β branch of the ves multigene family

Characterization of the Unusual Bidirectional ves Promoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family.

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform