Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

Semantic verbal fluency strategies in amnestic mild cognitive impairment.

OBJECTIVE: The primary aim of this study was to investigate the cognitive strategies used in semantic fluency (clustering, switching, and access to subcategories) in amnestic mild cognitive impairment (aMCI). In addition, we evaluated the impact of slowing in speed of information processing on semantic fluency performance. METHOD: Tests of semantic verbal fluency (animals, supermarket items) were administered to 33 participants with aMCI and 33 healthy older adults (HOA). A selected measure of speed of information processing was also administered. RESULTS: Analyses revealed significant impairment in word generation (animals, supermarket items) in the aMCI group relative to the HOA group (η² = .41). Furthermore, the aMCI group produced significantly smaller cluster sizes (η² = .12) and accessed fewer subcategories than the HOA group (η² = .11), whereas a difference in switching frequency between groups produced a small but nonsignificant effect. Although the aMCI group, as compared with the HOA group, demonstrated reductions in processing speed (η² = .17), covariance analyses adjusting for speed did not substantively alter the significant difference between groups in clustering and access to subcategories. CONCLUSIONS: When attempting semantic fluency, and as compared with healthy older adults, people with aMCI demonstrated difficulties in isolating semantic categories and loss of associative links within semantic categories. These findings are discussed in relation to an early degradation of semantic memory in aMCI.

Learning and memory in amnestic mild cognitive impairment: contribution of working memory.

In addition to deficits in delayed recall, recent research suggests that participants with amnestic mild cognitive impairment (aMCI) demonstrate diminished use of strategic encoding strategies during learning. Few studies have explored the cognitive mechanisms underlying this deficit. The aim of this study was to investigate in aMCI whether components of working memory (executive attention--attention set-shifting, dividing and focusing attention; and episodic buffer functions--strategic retrieval and manipulation of information) predict strategic encoding strategies during learning (semantic clustering). Thirty-three participants with aMCI and 33 healthy older adults (HOA) were administered neuropsychological tests assessing word-list learning and working memory. The aMCI group demonstrated significant impairment in acquisition, retrieval of information, and decreased use of semantic clustering strategies. Use of semantic clustering in the aMCI group was not predicted by measures of executive attention or phonemic verbal fluency, but was predicted by semantic verbal fluency performance. In the HOA group, semantic clustering was strongly related to semantic verbal fluency. These findings suggest that in aMCI, diminished strategic encoding strategies during learning (semantic clustering) is selectively related to the strategic function of the episodic buffer, but only when in interaction with the manipulation and retrieval of semantic associations.

Dynamics of intrapulmonary bacterial growth in a murine model of repeated microaspiration.

To study the change in intrapulmonary bacterial growth rate over time during Gram-negative pneumonia, a two-hit model of recurrent bacterial aspiration was developed in mice. A mutant of Klebsiella pneumoniae was isolated that could be distinguished from the wild type when cultured on appropriate media. These strains were intranasally administered, 4 h apart, to mice whose lungs were quantitatively cultured 24 h later. The relative burden of each aspirated inoculum was determined, and, using the administered dose and the number of bacteria from each inoculum present at the end of the experiment, first-order growth constants for each inoculum were calculated. Results indicate that after an initial aspiration of this organism, subsequently aspirated bacteria proliferate more slowly. When two aspirations occurred 4 h apart, the bacteria aspirated first represented 96% of total lung burden at 24 h. The growth constant of the second inoculum was related to the magnitude of the first inoculum in an inverse, nonlinear fashion. When parallel experiments were performed in complement C3-deficient mice, no suppression of the second inoculum was noted, suggesting that early upregulation of antibacterial activity in the lung is a C3-mediated event.

Development and application of a rat ovarian gene expression database.

The pituitary gonadotropins play a key role in follicular development and ovulation through the induction of specific genes. To identify these genes, we have constructed a genome-wide rat ovarian gene expression database (rOGED). The database was constructed from total RNA isolated from intact ovaries, granulosa cells, or residual ovarian tissues collected from immature pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin-treated rats at 0 h (no PMSG), 12 h, and 48 h post PMSG, as well as 6 and 12 h post human chorionic gonadotropin. The total RNA was used for DNA microarray analysis using Affymetrix Rat Expression Arrays 230A and 230B (Affymetrix, Santa Clara, CA). The microarray data were compiled and used for display of individual gene expression profiles through specially developed software. The final rOGED provides immediate analysis of temporal gene expression profiles for over 28,000 genes in intact ovaries, granulosa cells, and residual ovarian tissue during follicular growth and the preovulatory period. The accuracy of the rOGED was validated against the gene profiles for over 20 known genes. The utility of the rOGED was demonstrated by identifying six genes that have not been described in the rat periovulatory ovary. The mRNA expression patterns and cellular localization for each of these six genes (estrogen sulfotransferase, synaptosomal-associated protein 25 kDa, runt-related transcription factor, calgranulin B, alpha1-macroglobulin, and MAPK phosphotase-3) were confirmed by Northern blot analyses and in situ hybridization, respectively. The current findings demonstrate that the rOGED can be used as an instant reference for ovarian gene expression profiles, as well as a reliable resource for identifying important yet, to date, unknown ovarian genes.