Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Pressure induced structural changes and gas diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.

The role of *language in early childhood mathematics

Math literacy is imperative to succeed in society. Experience is key for acquiring math literacy. A preschooler's world is full of mathematical experiences. Children are continually counting, sorting and comparing as they play. As children are engaged in these activities they are using language as a tool to express their mathematical thinking. If teachers are aware of these teachable moments and help children bridge their daily experiences to mathematical concepts, math literacy may be enhanced. This study described the interactions between teachers and preschoolers, determining the extent to which teachers scaffold children's everyday language into expressions of mathematical concepts. Of primary concern were the teachers' responsive interactions to children's expressions of an implicit mathematical utterance made while engaged in block play. The parallel mixed methods research design consisted of two strands. Strand 1 of the study focused on preschoolers' use of everyday language and the teachers' responses after a child made a mathematical utterance. Twelve teachers and 60 students were observed and videotaped while engaged in block play. Each teacher worked with five children for 20 minutes, yielding 240 minutes of observation. Interaction analysis was used to deductively analyze the recorded observations and field notes. Using a priori codes for the five mathematical concepts, it was found children produced 2,831 mathematical utterances. Teachers ignored 60% of these utterances and responded to, but did not mediate 30% of them. Only 10% of the mathematical utterances were mediated to a mathematical concept. Strand 2 focused on the teacher's view of the role of language in early childhood mathematics. The 12 teachers who had been observed as part of the first strand of the study were interviewed. Based on a thematic analysis of these interviews three themes emerged: (a) the importance of a child's environment, (b) the importance of an education in society, and (c) the role of math in early childhood. Finally, based on a meta-inference of both strands, three themes emerged: (a) teacher conception of math, (b) teacher practice, and (c) teacher sensitivity. Implications based on the findings involve policy, curriculum, and professional development.

The relationship between the use of Academic Text Talk and the comprehension of scientific academic language for diverse second graders

Changing demographics impact our schools as children come from more linguistically and culturally diverse backgrounds. The various social, cultural, and economic backgrounds of the students affect their early language learning experiences which expose them to the academic language needed to succeed in school. Teachers can help students acquire academic language by introducing words that are within their Zone of Proximal Development and increasing exposure to and use of academic language. This study investigated the effects of increasing structured activities for students to orally interact with informational text on their scientific academic language development and comprehension of expository text. ^ The Academic Text Talk activities, designed to scaffold verbalization of new words and ideas, included discussion, retelling, games, and sentence walls. This study also evaluated if there were differences in scientific language proficiency and comprehension between boys and girls, and between English language learners and native English speakers. ^ A quasi-experimental design was used to determine the relationship between increasing students' oral practice with academic language and their academic language proficiency. Second graders (n = 91) from an urban public school participated in two science units over an 8 week period and were pre and post tested using the Woodcock Muñoz Language Survey-Revised and vocabulary tests from the National Energy Education Project. Analysis of covariance was performed on the pre to post scores by treatment group to determine differences in academic language proficiency for students taught using Academic Text Talk compared to students taught using a text-centered method, using the initial Florida Assessment for Instruction in Reading test as a covariate. Students taught using Academic Text Talk multimodal strategies showed significantly greater increases in their pre to posttest means on the Woodcock Muñoz Language Survey-Revised Oral Language Totals and National Energy Education Development Project Vocabulary tests than students taught using the text-centered method, ps < .05. Boys did not show significantly greater increases than girls, nor did English language learners show significantly greater increases than the native English speakers. ^ This study informs the field of reading research by evaluating the effectiveness of a multimodal combination of strategies emphasizing discourse to build academic language.^

Fiber Scaffolds of Poly (glycerol-dodecanedioate) and its Derivative via Electrospinning for Neural Tissue Engineering

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

Movement Effects on the Flow Physics and Nutrient Delivery in Engineered Valvular Tissues

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering (HVTE) owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by steady flow and scaffold flexure play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). In addition, scaffold flexure may enhance nutrient (e.g. oxygen, glucose) transport. In this study, we computationally quantified the i) magnitude of fluid-induced shear stresses; ii) the extent of temporal fluid oscillations in the flow field using the oscillatory shear index (OSI) parameter, and iii) glucose and oxygen mass transport profiles. Noting that sample cyclic flexure induces a high degree of oscillatory shear stress (OSS), we incorporated moving boundary computational fluid dynamic simulations of samples housed within a bioreactor to consider the effects of: 1) no flow, no flexure (control group), 2) steady flow-alone, 3) cyclic flexure-alone and 4) combined steady flow and cyclic flexure environments. We also coupled a diffusion and convention mass transport equation to the simulated system. We found that the coexistence of both OSS and appreciable shear stress magnitudes, described by the newly introduced parameter OSI-t , explained the high levels of engineered collagen previously observed from combining cyclic flexure and steady flow states. On the other hand, each of these metrics on its own showed no association. This finding suggests that cyclic flexure and steady flow synergistically promote engineered heart valve tissue production via OSS, so long as the oscillations are accompanied by a critical magnitude of shear stress. In addition, our simulations showed that mass transport of glucose and oxygen is enhanced by sample movement at low sample porosities, but did not play a role in highly porous scaffolds. Preliminary in-house in vitro experiments showed that cell proliferation and phenotype is enhanced in OSI-t environments.

Mother-Infant Reciprocity and the Development of Joint Attention in 12 Month Old Infants

Social contingency is the ability to connect social stimuli, such as those behaviors performed by oneself and those performed by others. Detecting social contingencies occurs by means of reciprocity through shared experiences with others. Reciprocity denotes a circumstance in which two individuals participate in a collaborative exchange, and is distinguished from an event in which two individuals engage in separate, unrelated activities. Specifically, reciprocity incorporates joint attention (JA), which occurs when two individuals simultaneously and visually attend to the same item. JA is facilitated by gazing and pointing, whereby one individual initiates the action and the second individual follows suit by, for example, gaze-following. However, little is known about the role the mother may play in the development of JA. The purpose of our study was to investigate social contingency between mothers and infants engaging in dyadic interactions. Thirty-three 12-month-old typically developing infants (M = 12.2, SD = .19; N = 19 males) were filmed for 10 minutes during free play with their mothers and toys provided by an experimenter. Reciprocity was measured by coding mother-infant interactions when a precise chain of events occurred: (1) mother initiated a bid by introducing a toy/activity or request to the infant, (2) infant accepted the bid/request by engaging in play with the given toy/activity, and (3) mother persisted by continuing to engage in play with said toy/activity. We computed a Pearson Correlation to assess the relation between the mothers’ initiations of JA and their infants’ responses to JA. We found a moderately positive correlation between the two variables (r= 0.37, p<.05). Our findings suggest that reciprocity, an important component of social relationships, during parent-infant dyads may serve as a scaffold for joint attention abilities, which have been linked to social and language development.