Technology in the Geometry Room

This study I am going to investigate the following research questions: 1 . Does the use of technology in a mathematics classroom result in better student performance? 2. Do students feel more motivated to do better in mathematics class when technology is used in the classroom? 3. Do students have positive attitudes and perceptions towards mathematics class when technology is used in the classroom

Investigation of possible causes for human-performance degradation during microgravity flight

The results of the first year of a three year study of the effects of microgravity on human performance are given. Test results show support for the hypothesis that the effects of microgravity can be studied indirectly on Earth by measuring performance in an altered gravitational field. The hypothesis was that an altered gravitational field could disrupt performance on previously automated behaviors if gravity was a critical part of the stimulus complex controlling those behaviors. In addition, it was proposed that performance on secondary cognitive tasks would also degrade, especially if the subject was provided feedback about degradation on the previously automated task. In the initial experimental test of these hypotheses, there was little statistical support. However, when subjects were categorized as high or low in automated behavior, results for the former group supported the hypotheses. The predicted interaction between body orientation and level of workload in their joint effect on performance in the secondary cognitive task was significant for the group high in automatized behavior and receiving feedback, but no such interventions were found for the group high in automatized behavior but not receiving feedback, or the group low in automatized behavior

Elucidation of the Rotavirus NSP4-Caveolin-1 and -Cholesterol Interactions Using Synthetic Peptides

Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4112–140 that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-12–20,  19–40 and C-caveolin-1161–180) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4112–140-N-caveolin-119–40 and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity

The Use of Bone Morphogenetic Protein in the Intervertebral Disk Space in Minimally Invasive Transforaminal Lumbar Interbody Fusion

Study Design: Retrospective Cohort. Objective: The objective of this study was to characterize one surgeon’s experience over a 10-year period using rhBMP-2 in the disk space for minimally invasive transforaminal lumbar interbody fusion (MIS TLIF). Summary of Background Data: MIS TLIF has been utilized as a technique for decreasing patients’ immediate postoperative pain, decreasing blood loss, and shortened hospital stays. Effectiveness and complications of rhBMP-2’s use in the disk space is limited because of its off-label status. Methods: Retrospective analysis of consecutive MIS TLIFs performed by senior author between 2004 and 2014. rhBMP-2 was used in the disk space in all cases. Patients were stratified based on the dose of rhBMP-2 utilized. Patients had 9 to 12 month computerized tomography scan to evaluate for bony fusion and continued follow-up for 18 months. Results: A total of 688 patients underwent a MIS TLIF. A medium kit of rhBMP-2 was utilized in 97 patients, and small kit was used in 591 patients. Fusion rate was 97.9% and this was not different between the 2 groups with 96/97 patients fusing in the medium kit group and 577/591 patients fusing in the small kit group. Five patients taken back to the operating room for symptomatic pseudoarthrosis, 4 reoperated for bony hyperostosis, and 10 radiographic pseudoarthroses that did not require reoperation. A statistically significant difference in the rate of foraminal hyperostosis was found when using a medium sized kit of rhBMP-2 was 4.12% (4/97 patients), compared with a small kit (0/591 patients, P=0.0004). Conclusions: Utilization of rhBMP-2 in an MIS TLIF leads to high fusion rate (97.9%), with an acceptable complication profile. The development of foraminal hyperostosis is a rare complication that only affected 0.6% of patients, and seems to be a dose related complication, as this complication was eliminated when a lower dose of rhBMP-2 was utilized

Mutational Analysis of the Rotavirus NSP4 Enterotoxic Domain that Binds to Caveolin-1

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction. Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP446-175-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study. Results: Mutations of the hydrophilic face (NSP446-175-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP446-175-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112 −140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea. Conclusions: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities

Secondary Structural and Functional Studies of Rotavirus NSP4 and Caveolin-1 Peptide-Peptide Interactions

The rotavirus NSP4 protein is the first described viral enterotoxin. Abundant data from our laboratory reveals that NSP4 binds both the N- and C-termini of caveolin- 1 (aa2-31 and 161-178, respectively). Yeast two-hybrid and peptide binding analysis mapped the caveolin-1 binding site to three hydrophobic residues within the amphipathic a-helix, enterotoxic peptide domain (aa114-135). The research studies herein utilized peptides to investigate the interaction between NSP4 and caveolin-1. Peptides were synthesized corresponding to the amphipathic a-helix and caveolin-1 binding domain of NSP4 (aa112-140) and to the N- (aa2-20 and 19-40) and C- (161-178) termini of caveolin-1, and were utilized in structural and functional studies. Fluorescence binding assays revealed that NSP4 (aa112-140) binds to the N-terminus (aa19-40) of caveolin-1 with a stronger affinity than the C-terminus (aa161-178). In addition, this assay further delineated the NSP4 binding domain on caveolin-1 to aa19-40. Secondary structural changes following NSP4-caveolin-1 peptide-peptide interactions were investigated by circular dichroism analysis. Changes in a-helix formation were observed only upon interaction of the NSP4112-140 peptide with the C-terminal caveolin-1 peptide (C-Cav161- 178). The NSP4112-140 peptide contains a potential cholesterol recognition amino acid consensus (CRAC) sequence. Therefore this peptide was examined for cholesterol binding. Results of the binding assay revealed NSP4 binds cholesterol with a Kd of 7.67 +/- 1.49nM and this interaction occurs via aa112-140. Mutation of amino acid residues within the CRAC motif resulted in weaker binding affinities between each of the corresponding mutant peptides and cholesterol. NSP4 peptides containing mutations within the hydrophobic and charged faces of the amphipathic a-helix, enterotoxic peptide and caveolin-1 binding domain of NSP4 were examined for changes in secondary structure as well as diarrhea induction in mouse pups. Circular dichroism analysis revealed that mutation of hydrophobic residues resulted in a decrease in a-helix formation, whereas mutation of acidic and basic charged residues caused little to no change in a-helical content. When tested for diarrhea induction in mouse pups, the peptides containing mutations of either the hydrophobic or basic charged residues did not cause diarrhea. Taken together, the results of this research suggest a complex interplay between NSP4 secondary structure, caveolin-1 and cholesterol binding and diarrheagenic function

A New N-terminal Recognition Domain in Caveolin-1 Interacts with Sterol Carrier Protein-2 (SCP-2)

Although plasma membrane domains, such as caveolae, provide an organizing principle for signaling pathways and cholesterol homeostasis in the cell, relatively little is known regarding specific mechanisms, whereby intracellular lipid-binding proteins are targeted to caveolae. Therefore, the interaction between caveolin-1 and sterol carrier protein-2 (SCP-2), a protein that binds and transfers both cholesterol and signaling lipids (e.g., phosphatidylinositides and sphingolipids), was examined by yeast two-hybrid, in vitro binding and fluorescence resonance energy transfer (FRET) analyses. Results of the in vivo and in vitro assays identified for the first time the N-terminal amino acids (aa) 1−32 amphipathic α helix of SCP-2 functionally interacted with caveolin-1. This interaction was independent of the classic caveolin-1 scaffolding domain, in which many signaling proteins interact. Instead, SCP-2 bound caveolin-1 through a new domain identified in the N-terminal domain of caveolin-1 between aa 34−40. Modeling studies suggested that electrostatic interactions between the SCP-2 N-terminal aa 1−32 amphipathic α-helical domain (cationic, positively charged face) and the caveolin-1 N-terminal aa 33−59 α helix (anionic, negatively charged face) may significantly contribute to this interaction. These findings provide new insights on how SCP-2 enhances cholesterol retention within the cell as well as regulates the distribution of signaling lipids, such as phosphoinositides and sphingolipids, at plasma membrane caveolae

Interview with Norman Krumholz

Norman Krumholz is a towering figure in the eyes of many planners. A proponent of equity planning—the term Paul Davidoff coined in 1965 to refer to planning for the whole city and prioritizing the needs of populations habitually excluded from the process—he pioneered approaches to improve the quality of life in disadvantaged communities. During Krumholz’s groundbreaking ten years as Cleveland’s planning director from 1969-1979, he put this theory into practice, later describing his experiences in a book entitled Making Equity Planning Work. Krumholz visited UNC-Chapel Hill on Oct. 9, 2008, to deliver a public lecture titled “New Roles and New Status for Planners.” In the talk, co-sponsored by the Department of City of Regional Planning and the Center for Urban and Regional Studies, Krumholz described how the changing shape of cities poses new challenges for today’s planners. He dispelled several myths about industrial cities, including the belief that “the decline can be reversed and the cities restored to their former glory.” Instead of emphasizing greening efforts, New Urbanism, and blockbuster stadium projects, he said planners should focus on the basics: fixing cities’ schools, services, and safety. In an exclusive Carolina Planning interview, editors Wendy Baucom and Heather Schroeder sat down with Krumholz before his public lecture to speak about the biggest issues facing today’s planners. Given his track record, it may be no surprise that Krumholz encourages planners to continue breaking the mold

Rotavirus NSP4: Cell Type-dependent Transport Kinetics to the Exofacial Plasma Membrane and Release from Intact Infected Cells

Background Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types. Methods Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls. Results Only full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells. Conclusions The intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study