The genome of Naegleria gruberi illuminates early eukaryotic versatility.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

Disc-associated proteins mediate the unusual hyperstability of the ventral disc in Giardia lamblia.

Giardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The 'cup-like' disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains - DAP5188KD and DAP6751KD -with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

Nucleophilic Thiol Proteins Bind Covalently to Abasic Sites in DNA.

In the course of studies on the enhancement of 1,2-dibromoethane-induced DNA base pair mutations by O6-alkylguanine-DNA alkyltransferase (AGT, MGMT), we discovered the facile reaction of AGT with an abasic site in DNA, leading to covalent cross-linking. The binding of AGT differs from the mechanism reported for the protein HMCES; instead it appears to involve formation of a stable thioglycoside. Facile cross-linking was also observed with the protease papain, which like AGT has a low pKa cysteine, and the tripeptide glutathione.

A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

The Food Web of Boiling Springs Lake Appears Dominated by the Heterolobosean Tetramitus thermacidophilus Strain BSL.

We studied the protist grazers of Boiling Springs Lake (BSL), an acid geothermal feature in Lassen Volcanic National Park, using a combination of culture and genetic approaches. The major predator in BSL is a vahlkampfiid ameba closely related (95% 18S+ITS rRNA identity) to Tetramitus thermacidophilus, a heterolobose ameboflagellate recently isolated from volcanic geothermal acidic sites in Europe and Russia, as well as an uncultured heterolobosean from the nearby Iron Mountain acid mine drainage site. Tetramitus thermacidophilus strain BSL is capable of surviving the physical extremes of BSL, with optimal growth at 38-50 °C and pH 2-5. This bacterivore also ingested conidiospores of the ascomycete Phialophora sp., but ultrastructural observations reveal the latter may not be readily digested, and conidia were not separable from the ameoboflagellate culture, suggesting a possible symbiosis. DGGE fingerprint transects studies showed the organism is restricted to near-lake environs, and we detected an average of ~500 viable cysts/cm(3) sediment on the shoreline. Other grazing protists were isolated from lakeshore environments, including the lobose amebae Acanthamoeba sp. and Hartmannella sp., and the kinetoplastid flagellate Bodo sp., but none could tolerate both low pH and high temperature. These appear to be restricted to cooler near lake geothermal features, which also contain other potential grazer morphotypes observed but not successfully cultured, including ciliates, euglenids, testate amebae, and possible cercozoans. We compare the food web of BSL with other acidic or geothermal sites, and discuss the impact of protists in this unique environment.

An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

Harnessing the power of new genetic tools to illuminate Giardia biology and pathogenesis.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

Blunted anticipatory stress responses on competition day in team sports athletes compared to individual sports athletes.

Knowledge of anticipatory stress responses before sports competitions is limited, thus this study examined the relationship between anticipatory stress in terms of salivary cortisol secretion in athletes on the morning of a competition and a comparison baseline day. Thirty-seven athletes collected three saliva samples over a 45-min period post-awakening (0, 30 and 45 min). Anticipatory stress was expressed as Area Under the Curve compared to ground (AUCg; total cortisol secretion). There was no significant difference in AUCg between baseline and competition days. However, a mixed two-factor ANOVA with day and sport type (individual vs. team) revealed a significant main effect of sport type (p < 0.01) and a significant interaction (p = 0.001). Individual athletes demonstrated increased AUCg on competition day compared to baseline, while team athletes demonstrated decreased AUCg on competition day compared to baseline. This blunting response was also observed when analysing the raw cortisol secretion levels upon awakening. These findings suggest there may be substantive differences in anticipatory stress between individual and team sport athletes.

ß-Variational autoencoders and transformers for reduced-order modelling of fluid flows.

Variational autoencoder architectures have the potential to develop reduced-order models for chaotic fluid flows. We propose a method for learning compact and near-orthogonal reduced-order models using a combination of a ß-variational autoencoder and a transformer, tested on numerical data from a two-dimensional viscous flow in both periodic and chaotic regimes. The ß-variational autoencoder is trained to learn a compact latent representation of the flow velocity, and the transformer is trained to predict the temporal dynamics in latent-space. Using the ß-variational autoencoder to learn disentangled representations in latent-space, we obtain a more interpretable flow model with features that resemble those observed in the proper orthogonal decomposition, but with a more efficient representation. Using Poincaré maps, the results show that our method can capture the underlying dynamics of the flow outperforming other prediction models. The proposed method has potential applications in other fields such as weather forecasting, structural dynamics or biomedical engineering.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Giardia flagellar motility is not directly required to maintain attachment to surfaces.

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined "suction-based" mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing "hydrodynamic model" of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.

The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment.

Giardia has unique microtubule structures, including the ventral disc, the primary organelle of attachment to the host, and the median body, a structure of undefined function. During attachment, the ventral disc has a domed conformation and enables Giardia to attach to the host intestinal epithelia within seconds. The mechanism of attachment via the ventral disc and the overall structure, function, and assembly of the ventral disc are not well understood. Our recent proteomic analysis of the ventral disc indicated that the median body protein (MBP), previously reported to localize exclusively to the median body, was primarily localized to the ventral disc. Using high-resolution light and electron microscopy, we confirm that the median body protein localizes primarily to the overlap zone of the ventral disc. The MBP also occasionally localized to the median body during prophase. To define the contribution of MBP to the ventral disc structure, we depleted MBP using an anti-MBP morpholino. We found that the ventral disc was no longer able to form properly and that the disc structure often had an aberrant nondomed or flattened horseshoe conformation. The ability of attached anti-MBP morpholino-treated trophozoites to withstand shear forces and normal forces was significantly decreased. Most notably, the plasma membrane contacts with the surface, including those of the bare area, were defective after the anti-MBP knockdown. To our knowledge, this is the first ventral disc protein whose depletion directly alters ventral disc structure, confirming that the domed ventral disc conformation is important for robust attachment.

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis.

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 um in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened crescent or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.

Affinity-purified Plasmodium tubulin provides a key reagent for antimalarial drug development.

Hirst et al. used a TOG-domain-based affinity-purification approach to reconstitute and define the in vitro dynamics of blood-stage Plasmodium falciparum αß-tubulin. This provides a key reagent for defining parasite microtubule (MT) dynamics and for evaluating the efficacy of anti-MT drugs throughout the complex parasite life cycle.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

An insider's guide to the microtubule cytoskeleton of Giardia.

Giardia intestinalis is a zoonotic, parasitic protist with a complex microtubule cytoskeleton critical for motility, attachment, intracellular transport, cell division and transitioning between its two life cycle stages - the cyst and the trophozoite. This review focuses on the structures of the primary elements of the microtubule cytoskeleton and cytoskeletal dynamics throughout this complex giardial life cycle. The giardial cytoskeleton has both highly dynamic elements and more stable MT structures, including several novel structures like the ventral disc that change conformation via unknown mechanisms. While our knowledge of the giardial cytoskeleton is primarily cytological, the completed Giardia genome and recently developed reverse genetic tools affords an opportunity to uncover the mechanisms of Giardia's cytoskeletal dynamics. Fundamental areas of giardial cytoskeletal biology remain to be explored, including high resolution imaging and compositional characterization of cytoskeletal structures required for elucidating the molecular mechanisms of cytoskeletal functioning.

Data-driven cardiovascular flow modelling: examples and opportunities.

High-fidelity blood flow modelling is crucial for enhancing our understanding of cardiovascular disease. Despite significant advances in computational and experimental characterization of blood flow, the knowledge that we can acquire from such investigations remains limited by the presence of uncertainty in parameters, low resolution, and measurement noise. Additionally, extracting useful information from these datasets is challenging. Data-driven modelling techniques have the potential to overcome these challenges and transform cardiovascular flow modelling. Here, we review several data-driven modelling techniques, highlight the common ideas and principles that emerge across numerous such techniques, and provide illustrative examples of how they could be used in the context of cardiovascular fluid mechanics. In particular, we discuss principal component analysis (PCA), robust PCA, compressed sensing, the Kalman filter for data assimilation, low-rank data recovery, and several additional methods for reduced-order modelling of cardiovascular flows, including the dynamic mode decomposition and the sparse identification of nonlinear dynamics. All techniques are presented in the context of cardiovascular flows with simple examples. These data-driven modelling techniques have the potential to transform computational and experimental cardiovascular research, and we discuss challenges and opportunities in applying these techniques in the field, looking ultimately towards data-driven patient-specific blood flow modelling.

Integrating multi-fidelity blood flow data with reduced-order data assimilation.

High-fidelity patient-specific modeling of cardiovascular flows and hemodynamics is challenging. Direct blood flow measurement inside the body with in-vivo measurement modalities such as 4D flow magnetic resonance imaging (4D flow MRI) suffer from low resolution and acquisition noise. In-vitro experimental modeling and patient-specific computational fluid dynamics (CFD) models are subject to uncertainty in patient-specific boundary conditions and model parameters. Furthermore, collecting blood flow data in the near-wall region (e.g., wall shear stress) with experimental measurement modalities poses additional challenges. In this study, a computationally efficient data assimilation method called reduced-order modeling Kalman filter (ROM-KF) was proposed, which combined a sequential Kalman filter with reduced-order modeling using a linear model provided by dynamic mode decomposition (DMD). The goal of ROM-KF was to overcome low resolution and noise in experimental and uncertainty in CFD modeling of cardiovascular flows. The accuracy of the method was assessed with 1D Womersley flow, 2D idealized aneurysm, and 3D patient-specific cerebral aneurysm models. Synthetic experimental data were used to enable direct quantification of errors using benchmark datasets. The accuracy of ROM-KF in reconstructing near-wall hemodynamics was assessed by applying the method to problems where near-wall blood flow data were missing in the experimental dataset. The ROM-KF method provided blood flow data that were more accurate than the computational and synthetic experimental datasets and improved near-wall hemodynamics quantification.

Draft Genome Sequence of the Free-Living, Iridescent Bacterium Tenacibaculum mesophilum Strain ECR.

Here, we report the genome sequence of Tenacibaculum mesophilum strain ECR, which was isolated from the river/ocean interface at Trunk River in Falmouth, Massachusetts. The isolation and sequencing were performed as part of the 2016 and 2018 Microbial Diversity courses at the Marine Biological Laboratory in Woods Hole, Massachusetts.