Recent advances in Cryptosporidium secreted proteins.

Recent advances in genetic manipulation of Cryptosporidium

Recent advances in genetic manipulation and genome sequencing have paved the way for a new generation of research organisms. The amphipod crustacean Parhyale hawaiensis is one such system. Parhyale are easy to rear and offer large broods of embryos amenable to injection, dissection, and live imaging. Foundational work has described Parhyale embryonic development, while advancements in genetic manipulation using CRISPR‐Cas9 and other techniques, combined with genome and transcriptome sequencing, have enabled its use in studies of arthropod development, evolution, and regeneration. This study introduces Parhyale development and life history, a catalog of techniques and resources for Parhyale research, and two case studies illustrating its power as a comparative research system.This article is categorized under:Comparative Development and Evolution > Evolutionary NoveltiesAdult Stem Cells, Tissue Renewal, and Regeneration > RegenerationComparative Development and Evolution > Model SystemsComparative Development and Evolution > Body Plan Evolution

Genetic manipulation is one of the central strategies that biologists use to investigate the molecular underpinnings of life and its diversity. Thus, advances in genetic manipulation usually lead to a deeper understanding of biological systems. During the last decade, the construction of chromosomes, known as synthetic genomics, has emerged as a novel approach to genetic manipulation. By facilitating complex modifications to chromosome content and structure, synthetic genomics opens new opportunities for studying biology through genetic manipulation. Here, we discuss different classes of genetic manipulation that are enabled by synthetic genomics, as well as biological problems they each can help solve.

Invasion of host cells by apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, is a multistep process. Central to invasion is the formation of a tight junction, an aperture in the host cell through which the parasite pulls itself before settling into a newly formed parasitophorous vacuole. Two protein groups, derived from different secretory organelles, the micronemal protein AMA1 and the rhoptry proteins RON2, RON4, and RON5, have been shown to form part of this structure, with antibodies targeting P. falciparum AMA1 known to inhibit invasion, probably via disruption of its association with the PfRON proteins. Inhibitory AMA1-binding peptides have also been described that block P. falciparum merozoite invasion of the erythrocyte. One of these, R1, blocks invasion some time after initial attachment to the erythrocyte and reorientation of the merozoite to its apical pole. Here we show that the R1 peptide binds the PfAMA1 hydrophobic trough and demonstrate that binding to this region prevents its interaction with the PfRON complex. We show that this defined association between PfAMA1 and the PfRON complex occurs after reorientation and engagement of the actomyosin motor and argue that it precedes rhoptry release. We propose that the formation of the AMA1-RON complex is essential for secretion of the rhoptry contents, which then allows the establishment of parasite infection within the parasitophorous vacuole.

Decoding the microbiome: advances in genetic manipulation for gut bacteria.

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.

Environmental stresses are the most significant limiting factors, posing severe threats to agricultural production worldwide. Abiotic stress factors, including drought, salinity, water-logging, temperature extremes (heat, freezing, and chilling), herbicides, and high heavy metals, reduce global annual food production by more than 50%. To address these issues, most important strategies like conventional breeding and genetic engineering have been applied to increase abiotic stress tolerance in agricultural crops. Researchers have established diverse genetic manipulation techniques, such as transgenic approach, RNA interference, and CRISPR/Cas9 technology, which encompass the promise to boost agricultural crops under abiotic stress circumstances. Scientists have identified several key genes and transcription factors associated with stress responses, generally those are played in osmotic-pressure regulation, antioxidant defense mechanism, and stress-responsive signaling pathways, and these can achieve through over-expression method, silencing approaches, and knockout technology. These advances in genetic manipulation not only enhance crop endurance under stress but also contribute to sustainable agriculture by decreasing the requirement of chemical fertilizers. However, challenges remain, together with regulatory hurdles, public acceptance, and the need for wide-ranging field testing to assess the long-term impact of genetically modified crops. As research progresses, the augmentation of genetic engineering methods may possibly modernize agriculture, making it more resilient to the challenges of a quickly altering the climatic condition. This chapter will underline that how genetic manipulation of crops enables them to cope with abiotic stress tolerance.

Advances in genetic manipulation by gene targeting and introduction of transgenes have necessitated the development of arterial injury models for the mouse. Using genetically altered mice in these models would allow researchers to define the role of certain genes in the events associated with intimal lesion formation, remodeling, and endothelial cell growth. For the rat, the balloon catheter denudation model has become the most widely applied model for the study of these events. Adopting this balloon catheter injury model for the mouse carotid artery has presented a major technical challenge, largely because there is no commercially available catheter small enough to use with mice. In our search for other suitable devices that would achieve complete endothelial denudation, we developed the guide wire denudation model (1) and later, the technically less challenging ligation model that causes intimal hyperplasia in the absence of widespread endothelial denudation (2). These two models are described in detail in this chapter.

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.

The obligate intracellular apicomplexan parasite Neospora caninum (N. caninum) is closely related to Toxoplasma gondii (T. gondii). The dense granules, which are present in all apicomplexan parasites, are important secretory organelles. Dense granule (GRA) proteins are released into the parasitophorous vacuole (PV) following host cell invasion and are known to play important roles in the maintenance of the host-parasite relationship and in the acquisition of nutrients. Here, we provide a detailed characterization of the N. caninum dense granule protein NcGRA9. The in silico genomic organization and key protein characteristics are described. Immunofluorescence-based localization studies revealed that NcGRA9 is located in the dense granules and is released into the interior of the PV following host cell invasion. Immunogold-electron microscopy confirmed the dense granule localization and showed that NcGRA9 is associated with the intravacuolar network. In addition, NcGRA9 is found in the “excreted secreted antigen” (ESA) fraction of N. caninum. Furthermore, by analysing the distribution of truncated versions of NcGRA9, we provide evidence that the C-terminal region of this protein is essential for the targeting of NcGRA9 into the dense granules of N. caninum, and the truncated proteins show reduced secretion.

Waaland, J. R.1 & Stiller, J. W.2 1Department of Botany, University of Washington, Seattle, WA 98195 USA; 2Department of Biology, East Carolina University, Greenville, NC 27858 USAMacroalgae are important components of aquatic ecosystems. Some are harvested or cultivated for economic uses while others are of interest for their phylogenetic or systematic positions. Although most genes known from macroalgae have been isolated for comparative evolutionary analysis, some have been the subject of more detailed molecular investigations. We examine the current state of knowledge for several macroalgae as candidates for genomic study. Selection criteria for target taxa include features such as a well known sexual life history, availability of established laboratory cultures, mutant strains, basic genetic studies, fossil records, and ecological and economic importance. Among the algae to be considered are: Porphyra, Gracilaria, Ectocarpus, Macrocystis, Laminaria, Fucus, Ulva, Chara and Nitella. A strong case can be made for each of these taxa; however, we will emphasize Porphyra yezoensis because of its importance as a food source, its well‐characterized and easily manipulated reproductive biology, its relatively small genome size, and recent technical advances in genetic manipulation that should lead to fruitful exploitation of genomic information as it becomes available. Further, the genome of a red alga is an attractive target for comparison with those of other multicellular eukaryotes that have been the object of sequencing projects thus far.

The first reproductively viable genetically modified mice were created in 1982 by Richard Palmiter and Ralph Brinster (Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300: 611-615). In the subsequent 30 plus years, numerous ground-breaking technical advancements in genetic manipulation have paved the way for improved spatially and temporally targeted research. Molecular genetic studies have been especially useful for probing the molecules and circuits underlying how organisms learn and remember—one of the most interesting and intensively investigated questions in neuroscience research. Here, we discuss selected genetic tools, focusing on corticohippocampal circuits and their implications for understanding learning and memory.

Combining genetic and genomic approaches to study mood disorders

For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post-translational modifications, particularly glycosylation, which unlike protein synthesis, is a non-templated process. Consequently, both native and recombinant glycoprotein production generate heterogeneous mixtures containing variable amounts of different glycoforms. Stability, potency, plasma half-life, and immunogenicity of the glycoprotein biologic are directly influenced by the glycoforms. Recently, CHO cells have also been explored for production of therapeutic glycosaminoglycans (e.g., heparin), which presents similar challenges as producing glycoproteins biologics. Approaches to controlling heterogeneity in CHO cells and directing the biosynthetic process toward desired glycoforms are not well understood. A systems biology approach combining different technologies is needed for complete understanding of the molecular processes accounting for this variability and to open up new venues in cell line development. In this review, we describe several advances in genetic manipulation, modeling, and glycan and glycoprotein analysis that together will provide new strategies for glycoengineering of CHO cells with desired or enhanced glycosylation capabilities.

In vivo research is critical to the functional dissection of
\nmulti-organ systems and whole organism physiology, and
\nthe laboratory mouse remains a quintessential animal model
\nfor studying mammalian, especially human, pathobiology.
\nEnabled by technological innovations in genome sequencing,
\nmutagenesis and genome editing, phenotype analyses, and
\nbioinformatics, in vivo analysis of gene function and dysfunction
\nin the mouse has delivered new understanding of the
\nmechanisms of disease and accelerated medical advances.
\nHowever, many significant hurdles have limited the elucidation
\nof mechanisms underlying both rare and complex,
\nmultifactorial diseases, leaving significant gaps in our scientific
\nknowledge. Future progress in developing a functionally
\nannotated genome map depends upon studies in model organisms,
\nnot least the mouse