Issues in Action Formation : Requests and the Problem with x

In previous interactional studies of formats for utterances doing requests, attention has been given to the initial verb (such as can/could or wonder) and possibly the subject (especially I vs you). The current study examines the main types of grammatical variation found in what we call the " x component," that is the segment after the initial verb and subject. We examine two types of requests: those with can you x and those with wonder x, and we find that variations in the x component in these requests are associated with variations in the unfolding development of the request sequences. We thus suggest that the x component is crucial to the interactional work accomplished by the requesting utterance.Peer reviewe

Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

The orotidine-5\u27-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting DeltaOMPDC, DeltaUP, and DeltaOMPDC DeltaUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The DeltaUP knockout strain was replication competent and virulent. In contrast, the DeltaOMPDC and DeltaOMPDC DeltaUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the DeltaOMPDC strain but not the DeltaOMPDC DeltaUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the DeltaOMPDC and DeltaOMPDC DeltaUP knockout strains exhibited extreme attenuation in murine virulence (approximately 8 logs). Genetic complementation of the DeltaOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated DeltaOMPDC strain or the DeltaOMPDC DeltaUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting DeltaOMPDC and DeltaOMPDC DeltaUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered

Mission San Jose Repointing and Underpinning Project, San Antonio, Texas

This report contains the results of archaeological work performed by the Center for Archaeological Research (CAR) for the National Park Service (NPS) under Contract Numbers: 1443PX7600-97053 and 1443PX760098028. Both projects were carried out under Texas Historical Commission Permit Number 1841. The bulk of the report deals with the results of shovel testing and archaeological excavations conducted as part of the Indian Quarters Stabilization project at Mission San Jose y San Miguel de Aguayo (41BX3) for the San Antonio Missions National Historical Park. Appendix 1 of this report contains the results of shovel testing and the monitoring of sign removal and installation conducted at Mission San Juan, Mission Espada, Mission Concepcion, and Mission San Jose as part of the Missions Signage Removal/lnstallation Project. Mission San Jose is located ca. seven miles south of downtown San Antonio on a high terrace overlooking the west bank of the San Antonio River. On October 27 and 29, 1997, CAR personnel excavated a total of 3 9 shovel tests (ST) along the southern, eastern, and western outer walls of the mission. The test excavations were conducted in preparation for grade alterations to be undertaken along the exterior and interior walls of the mission compound. In June and late July 1998, CAR personnel conducted excavations outside the south wall and within Room 54 adjacent to the western comer of the southeast gate of the mission. These excavations were undertaken to mitigate the impact of underpinning efforts on the southwest comer of the Southeast Gate of the mission being undertaken by the NPS through a major construction contract. The shovel testing and excavations showed that: 1) some high density artifact concentrations are present outside of the western and eastern mission walls possibly representing colonial middens; 2) a large portion of the soils and cultural materials found immediately adjacent the south wall of the mission show signs of disturbance from the Civil Works Administration (CWA) efforts to relocate the colonial foundation and outer walls of the mission; 3) much of the cultural material-bearing matrix found along the south wall of the mission, inside Room 54, is also disturbed to a depth of approximately 19 inches bs, and 4) a colonial living surface exists immediately below the disturbed zone in portions of the interior of Room 54 and under the southeast gate. Due to the urgency of the construction contractor to complete the regrading and underpinning efforts to reduce erosion and stabilize the southwest comer of the southeast gate, work on these projects proceeded concurrently and immediately following the archaeological investigations. However, it is the opinion of CAR that the regrading project impacted no intact cultural materials and, in fact, may have served to preserve, through burial, future disturbances to the deeper buried (less disturbed) colonial zone. In addition, the archaeological investigations conducted by CAR have recovered significant data and mitigated the impact of construction activities related to the underpinning of the southwest comer of the southeast gate. We commend the NPS and the construction contractor for their cooperation and collaboration in these efforts

An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype

Archaeological Testing for the Mission Road Realignment Project, Phase II, at Mission Concepcion, San Antonio, Texas

In July 1988, the Center for Archaeological Research (CAR) contracted with the city of San Antonio to perform archaeological testing for the Mission Road Realignment Project. This project was designated as Phase II since CAR performed previous archaeological testing during February 1987 (Labadie 1989). The Mission Road Realignment Project, Phase II proposed to relocate the position of Mission Road outside the line of the original west wall of Mission Concepcion. The testing sought to determine whether any structural remains or cultural deposits that may have been located outside the mission wall would be impacted by the proposed roadway. Archaeological testing with hand-excavated units and backhoe trenches established the location of the west wall of the mission quadrangle and a portion of an interior structure wall foundation with an associated hearth and cultural midden. The northwest corner of the mission is believed to be located under the current Mission Road. Mission-period pottery, metals tools, projectile points, and animal bone were recovered from the excavations

Perception of fonts: Perceived personality traits and uses

Often credited with creating first impressions, fonts are typically classified according to unique typographical features (serif, sans serif, etc) and overall appearance. The combination of appearance and typographical features often lead graphic artists and typographers to describe typefaces using personality traits (.less cuddly, more assertive, Berry, 2004). In a BBC audio program (Peacock, 2005), fonts were depicted as feminine and masculine, among other traits. Feminine fonts were described as fine, serifed, sleek, and elegant; masculine fonts were characterized as being blocky and bold. This study sought to determine if certain personalities and uses are associated with various fonts. Using an online survey, participants rated the personality of 20 fonts using 15 adjective pairs. In addition, participants viewed the same 20 fonts and selected which uses were most appropriate. Results suggested that personality traits are indeed attributed to fonts based on their design family (Serif, Sans-Serif, Modern, Monospace, Script/Funny) and are associated with appropriate uses. Implications of these results to the design of online materials and websites are discussed

Type II Toxoplasma Gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8+ T cell epitopes that elicit corresponding antigen-specific CD8+ T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8+ T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development since brain tissue cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission

CD4 T-Cell Suppression by Cells from Toxoplasma gondii-Infected Retinas Is Mediated by Surface Protein PD-L1

In the inflamed retina, CD4(+) T cells can cause retinal damage when they are not properly regulated. Since tissue expression of major histocompatibility complex (MHC) class II and costimulatory molecules is a key mechanism for regulating effector T cells, we tested the hypothesis that upregulation of these proteins in the retina contributes to the regulation of CD4 T cells. Here we report that in retinas infected with the protozoan parasite Toxoplasma gondii, MHC class II is upregulated on infiltrating leukocytes as well as on resident retinal cells, including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members (CD80, CD86, ICOS-L, and programmed death ligand 2 [PD-L2]) were not expressed on class II(+) cells. In contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and resident retinal MHC class II-expressing cells. Retinal cells from Toxoplasma-infected animals were able to suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that the expression of MHC class II and PD-L1 was critically dependent on gamma interferon (IFN-gamma) expression. These data suggest that retinal MHC class II and PD-L1 expression is a novel mechanism by which the retina protects itself from CD4 T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses

Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii, disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil aux- otrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH of T. gondii (Tg DHODH) failed. To further address the essentiality of Tg DHODH, mutant gene alleles deficient in Tg DHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient Tg DHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show that Tg DHODH is essential for the synthesis of pyrimidines and suggest that Tg DHODH is required for a second essential function independent of its role in pyrimidine biosynthesis

The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence

Toxoplasma gondii infects up to one third of the world\u27s population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms