Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii

ABSTRACT Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG I is plasma membrane associated whereas PKG II is cytosolic in Toxoplasma gondii . To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii . By combining AID regulation with genome editing strategies, we determined that PKG I is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG II is functionally insufficient and dispensable in the presence of PKG I . The difference in functionality mapped to the first 15 residues of PKG I , containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii . IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii . We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii . </jats:p

A retrospective segmentation analysis of placental volume by magnetic resonance imaging from first trimester to term gestation

Background Abnormalities of the placenta affect 5–7% of pregnancies. Because disturbances in fetal growth are often preceded by dysfunction of the placenta or attenuation of its normal expansion, placental health warrants careful surveillance. There are limited normative data available for placental volume by MRI. Objective To determine normative ranges of placental volume by MRI throughout gestation. Materials and methods In this cross-sectional retrospective analysis, we reviewed MRI examinations of pregnant females obtained between 2002 and 2017 at a single institution. We performed semi-automated segmentation of the placenta in images obtained in patients with no radiologic evidence of maternal or fetal pathology, using the Philips Intellispace Tumor Tracking Tool. Results Placental segmentation was performed in 112 women and had a high degree of interrater reliability (single-measure intraclass correlation coefficient =0.978 with 95% confidence interval [CI] 0.956, 0.989; P<0.001). Normative data on placental volume by MRI increased nonlinearly from 6 weeks to 39 weeks of gestation, with wider variability of placental volume at higher gestational age (GA). We fit placental volumetric data to a polynomial curve of third order described as placental volume = –0.02*GA3 + 1.6*GA2 – 13.3*GA + 8.3. Placental volume showed positive correlation with estimated fetal weight (P=0.03) and birth weight (P=0.05). Conclusion This study provides normative placental volume by MRI from early first trimester to term gestation. Deviations in placental volume from normal might prove to be an imaging biomarker of adverse fetal health and neonatal outcome, and further studies are needed to more fully understand this metric. Assessment of placental volume should be considered in all routine fetal MRI examinations

Prediction-Based Learning and Processing of Event Knowledge.

Knowledge of common events is central to many aspects of cognition. Intuitively, it seems as though events are linear chains of the activities of which they are comprised. In line with this intuition, a number of theories of the temporal structure of event knowledge have posited mental representations (data structures) consisting of linear chains of activities. Competing theories focus on the hierarchical nature of event knowledge, with representations comprising ordered scenes, and chains of activities within those scenes. We present evidence that the temporal structure of events typically is not well-defined, but it is much richer and more variable both within and across events than has usually been assumed. We also present evidence that prediction-based neural network models can learn these rich and variable event structures and produce behaviors that reflect human performance. We conclude that knowledge of the temporal structure of events in the human mind emerges as a consequence of prediction-based learning

Punishing an error improves learning: The influence of punishment magnitude on error-related neural activity and subsequent learning

Punishing an error to shape subsequent performance is a major tenet of individual and societal level behavioral interventions. Recent work examining error-related neural activity has identified that the magnitude of activity in the posterior medial frontal cortex (pMFC) is predictive of learning from an error, whereby greater activity in this region predicts adaptive changes in future cognitive performance. It remains unclear how punishment influences error-related neural mechanisms to effect behavior change, particularly in key regions such as pMFC, which previous work has demonstrated to be insensitive to punishment. Using an associative learning task that provided monetary reward and punishment for recall performance, we observed that when recall errors were categorized by subsequent performance— whether the failure to accurately recall a number–location association was corrected at the next presentation of the same trial—the magnitude of error-related pMFC activity predicted future correction. However, the pMFC region was insensitive to the magnitude of punishment an error received and it was the left insula cortex that predicted learning from the most aversive outcomes. These findings add further evidence to the hypothesis that error-related pMFC activity may reflect more than a prediction error in representing the value of an outcome. The novel role identified here for the insular cortex in learning from punishment appears particularly compelling for our understanding of psychiatric and neurologic conditions that feature both insular cortex dysfunction and a diminished capacity for learning from negative feedback or punishment. Copyright©2010 the author

Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9

ABSTRACT Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excel-lent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special ge-netic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 con-tributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity. IMPORTANCE Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. How-ever, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to suc

Solving Defender-Attacker-Defender Models for Infrastructure Defense

In Operations Research, Computing, and Homeland Defense, R.K. Wood and R.F. Dell, editors, INFORMS, Hanover, MD, pp. 28-49.The article of record as published may be located at http://dx.doi.org10.1287/ics.2011.0047This paper (a) describes a defender-attacker-defender sequential game model (DAD) to plan defenses for an infrastructure system that will enhance that system's resilience against attacks for an intelligent adversary, (b) describes a realistic formulation of DAD for defending a transportation network, (c) develops a decomposition algorithm for solving this instance of DAD and others, and (d) demonstrates the solution of a small transportation-network example. A DAD model generally evaluates system operation through the solution of an optimization model, and the decomposition algorithm developed here requires only that this system-operation model be continuous and convex. For example, our transportation-network example incorporates a congestion model with a (convex) nonlinear objective function and linear constraints

Elemental signatures in otoliths of larval walleye pollock (Theragra chalcogramma) from the northeast Pacific Ocean

The contents of the Fishery Bulletin are not been copyrighted. The definitive version was published in Fishery Bulletin 102 (2004): 604-616.The objectives of this study are to determine if larval walleye pollock from different geographic localities can be distinguished based on elemental signatures in their otoliths. By analyzing sagittal otoliths with both EPMA and laser ablation ICP-MS, we hoped to identify greater differences among locations than would have been possible by using either technique in isolation. If successful, the study may provide a powerful tool for determining stock structure and tracing migration pathways of walleye pollock in the north Pacific. These data could then be used by managers of one of the world's largest single species fisheries to direct the sustainable harvest of this considerable natural resource.This work was funded by North Pacific Marine Research Program to KMB, SRT and KPS, and was supported in part by NSF grants OCE-9871047 and OCE-0134998 to SRT