In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis.

UnlabelledToxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.ImportanceMost intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease

Novel components of the Toxoplasma inner membrane complex revealed by BioID.

UNLABELLED:The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE:The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma

Analysis of Lunar Boulder Tracks: Implications for Trafficability of Pyroclastic Deposits

In a new era of lunar exploration, pyroclastic deposits have been identified as valuable targets for resource utilization and scientific inquiry. Little is understood about the geomechanical properties and the trafficability of the surface material in these areas, which is essential for successful mission planning and execution. Past incidents with rovers highlight the importance of reliable information about surface properties for future, particularly robotic, lunar mission concepts. Characteristics of 149 boulder tracks are measured in Lunar Reconnaissance Orbiter Narrow Angle Camera images and used to derive the bearing capacity of pyroclastic deposits and, for comparison, mare and highland regions from the surface down to ~5‐m depth, as a measure of trafficability. Results are compared and complemented with bearing capacity values calculated from physical property data collected in situ during Apollo, Surveyor, and Lunokhod missions. Qualitative observations of tracks show no region‐dependent differences, further suggesting similar geomechanical properties in the regions. Generally, bearing capacity increases with depth and decreases with higher slope gradients, independent of the type of region. At depths of 0.19 to 5 m, pyroclastic materials have bearing capacities equal or higher than those of mare and highland material and, thus, may be equally trafficable at surface level. Calculated bearing capacities based on orbital observations are consistent with values derived using in situ data. Bearing capacity values are used to estimate wheel sinkage of rover concepts in pyroclastic deposits. This study's findings can be used in the context of traverse planning, rover design, and in situ extraction of lunar resources

Using Boulder Tracks as a Tool to Understand the Bearing Capacity of Permanently Shadowed Regions of the Moon

Permanently shadowed regions (PSRs) are abundant at the lunar poles. They experience no direct sunlight and reach temperatures as low as 30 K. PSRs are of interest as evidence suggests that some may contain water ice (H2O/OH‐), which could provide a record of the evolution of volatiles in the inner solar system. This water ice is also a critical resource for life‐support systems and rocket propellant. A better understanding of mechanical properties of PSR regolith, such as its bearing capacity, will help optimize the design of future exploration rovers and landers. Thirteen boulder tracks were identified on the edge of, or inside, south polar lunar PSR enhanced imagery and used to estimate the strength of the PSR regolith at latitudes of 70° to 76° in sites with maximum annual temperatures of 65 to 210 K. PSR boulder track features are similar to those observed in highland, mare, and pyroclastic regions of the Moon, implying similar properties of the regolith. Measured features were used to estimate bearing capacity for PSR regolith at depths of ~0.28 to 4.68 m. Estimated bearing capacity values suggest that these PSRs may be somewhat stronger than highland and mare regions at depths of 0.28 to 1.00 m. Bearing capacity in these PSRs is statistically the same as those in other regions of the Moon at depths of 1.00 to 2.00 m. The results of this study can be used to infer bearing capacity as one measure for the trafficability of lower‐latitude PSRs of the type measured here

Study of γπ→ππ\gamma\pi \to \pi\pi below 1 GeV using Integral Equation Approach

The scattering of $\gamma \pi \to \pi \pi$ is studied using the axial anomaly, elastic unitarity, analyticity and crossing symmetry. Using the technique to derive the Roy's equation, an integral equation for the P-wave amplitude is obtained in terms of the strong P-wave pion pion phase shifts. Its solution is obtained numerically by an iteration procedure using the starting point as the solution of the integral equation of the Muskelshsvilli-Omnes type. It is, however, ambiguous and depends sensitively on the second derivative of the P-wave amplitude at $s=m_\pi^2$ which cannot directly be measured.Comment: 26 pages, 10 figure

Caenorhabditis elegans Show Preference for Stimulants and Potential as a Model Organism for Medications Screening

The nematode Caenorhabditis elegans (C. elegans) is a popular invertebrate model organism to study neurobiological disease states. This is due in part to the intricate mapping of all neurons and synapses of the entire animal, the wide availability of mutant strains, and the genetic and molecular tools that can be used to manipulate the genome and gene expression. We have shown that, C. elegans develops a conditioned preference for cues that had previously been paired with either cocaine or methamphetamine exposure that is dependent on dopamine neurotransmission, similar to findings using place conditioning with rats and mice. In the current study, we show C. elegans also display a preference for, and self-exposure to, cocaine and nicotine. This substance of abuse (SOA) preference response can be selectively blocked by pretreatment with naltrexone and is consistent with the recent discovery of an opioid receptor system in C. elegans. In addition, pre-exposure to the smoking cessation treatment varenicline also inhibits self-exposure to nicotine. Exposure to concentrations of treatments that inhibit SOA preference/self-exposure did not induce any significant inhibition of locomotor activity or affect food or benzaldehyde chemotaxis. These data provide predictive validity for the development of high-throughput C. elegans behavioral medication screens. These screens could enable fast and accurate generation of data to identify compounds that may be effective in treating human addiction. The successful development and validation of such models would introduce powerful and novel tools in the search for new pharmacological treatments for substance use disorders, and provide a platform to study the mechanisms that underlie addictions