Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method.

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5' side of the modification site. This 5' binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure-activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5' binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure-function relationships in ADAR reactions

DNA editing in DNA/RNA hybrids by adenosine deaminases that act on RNA.

Adenosine deaminases that act on RNA (ADARs) carry out adenosine (A) to inosine (I) editing reactions with a known requirement for duplex RNA. Here, we show that ADARs also react with DNA/RNA hybrid duplexes. Hybrid substrates are deaminated efficiently by ADAR deaminase domains at dA-C mismatches and with E to Q mutations in the base flipping loop of the enzyme. For a long, perfectly matched hybrid, deamination is more efficient with full length ADAR2 than its isolated deaminase domain. Guide RNA strands for directed DNA editing by ADAR were used to target six different 2΄-deoxyadenosines in the M13 bacteriophage ssDNA genome. DNA editing efficiencies varied depending on the sequence context of the editing site consistent with known sequence preferences for ADARs. These observations suggest the reaction within DNA/RNA hybrids may be a natural function of human ADARs. In addition, this work sets the stage for development of a new class of genome editing tools based on directed deamination of 2΄-deoxyadenosines in DNA/RNA hybrids

The neural correlates of speech motor sequence learning

Speech is perhaps the most sophisticated example of a species-wide movement capability in the animal kingdom, requiring split-second sequencing of approximately 100 muscles in the respiratory, laryngeal, and oral movement systems. Despite the unique role speech plays in human interaction and the debilitating impact of its disruption, little is known about the neural mechanisms underlying speech motor learning. Here, we studied the behavioral and neural correlates of learning new speech motor sequences. Participants repeatedly produced novel, meaningless syllables comprising illegal consonant clusters (e.g., GVAZF) over 2 days of practice. Following practice, participants produced the sequences with fewer errors and shorter durations, indicative of motor learning. Using fMRI, we compared brain activity during production of the learned illegal sequences and novel illegal sequences. Greater activity was noted during production of novel sequences in brain regions linked to non-speech motor sequence learning, including the BG and pre-SMA. Activity during novel sequence production was also greater in brain regions associated with learning and maintaining speech motor programs, including lateral premotor cortex, frontal operculum, and posterior superior temporal cortex. Measures of learning success correlated positively with activity in left frontal operculum and white matter integrity under left posterior superior temporal sulcus. These findings indicate speech motor sequence learning relies not only on brain areas involved generally in motor sequencing learning but also those associated with feedback-based speech motor learning. Furthermore, learning success is modulated by the integrity of structural connectivity between these motor and sensory brain regions.R01 DC007683 - NIDCD NIH HHS; R01DC007683 - NIDCD NIH HH

Judicial Recognition of Hospital Independent Duty of Care to Patients: Hannola v. City of Lakewood

This Note will examine the court\u27s rationale in Hannola v City of Lakewood and the previous leading Ohio case on emergency room care, Cooper v. Sisters of Charity. This Note will similarly examine control tests of employment, the concept of apparent authority and the series of cases on independent duty of care which have been decided in the eleven years between Cooper and Hannola. It is the conclusion of this Note that the Hannola decision is more consistent with the realities of employment and service in the health care industry, and that the public policy arguments presented by the court require significant modifications in decisional rationale for medical negligence cases

'The task of Fort Hare in terms of the Transkei and Ciskei': educational planning at Fort Hare in the 1960's

Paper presented at the Wits History Workshop: Structure and Experience in the Making of Apartheid, 6-10 February, 1990This paper examines government intervention at Fort Hare after 1960. The first part of the paper will examine how the Department of Bantu Education and the new administrators of the college tried to transform Fort Hare into an institution where students were prepared, in terms both of skill and attitude, for a political future in the homelands; the second part of the paper accesses the success of this project, by examining courses of study chosen by students, their pass rates and where graduates were employed

How Does Goal Setting Impact Intrinsic Motivation And Does It Help Lead To Enhanced Learning At The Kindergarten Level?

The research question addressed in this project was, how does goal setting impact intrinsic motivation and does it help lead to enhanced learning at the kindergarten level? It documents one teacher’s creation of letter identification pre and post-tests along with check points in between the pre and post-tests. She utilized student graphing charts to document student progress. It integrates Frank’s seven-step process on how to set a goal for yourself. It also discusses Locke’s theories for goal setting and Deci & Ryan’s SelfDetermination Theory. The author documents results collected based on the different tests to help validate the study. She describes both success and struggles in implementing the study and concludes that her findings agree with those of previous researchers

Contaminant migration of oil-and-gas drilling fluids within the glaciated sediments of north-central North Dakota

A common practice during oil and gas well-site reclamation in North Dakota is to bury the drilling muds in shallow trenches near the borehole. These muds are saltwater based (between 100,000 and 300,000 mg/L of NaCl) and can contain high concentrations of chromium, lead, and other. toxic trace metals. Two reclaimed oil and gas well sites were chosen for study in north-central North Dakota: the Winderl site in southeastern Renville County, and the Fossum site in west-central Bottineau County. The Winderl oil well was drilled in 1959, and the drilling fluids were disposed of in a shallow pit excavated in Pleistocene glaciofluvial deposits. The Fossum oil well was drilled in 1978 and the drilling fluids were disposed of in trenches excavated in Pleistocene till. A total of 41 shallow piezometers (maximum depth is 62 feet {18.9 m)) and 13 pressure-vacuum lysimeters were installed in and around the two disposal sites to obtain groundwater and pore-water samples. Vertical electrode sounding resistivity profiles were conducted at both sites utilizing 14 electrode spacings down to a depth of 100 feet (30.5 m). Sediment samples were obtained with Shelby tubes for x-ray fluorescence and x-ray diffraction analyses. Additional chemical analyses were performed on saturated-paste extracts from the Shelby-tube samples. The results of chemical analyses of pore water, groundwater, saturated-paste extracts, and the earth resistivity surveys indicate that leachate is being generated from buried drilling fluid at both study sites. At the Winderl site, contaminants have migrated beyond 400 feet (122 m), the extent of the monitored area, which has resulted in degradation of the Spring Coulee Creek Aquifer. A one-dimensional analytical solute transport equation was utilized to illustrate the potential for contaminant migration at the site. The equation predicts high concentration of contaminants over 3300 feet (1000 m) from the source area. Contaminant migration within the till at the Fossum site is believed to occur along fractures directly below the water table. The estimated groundwater velocity through these fractures is 3.8 m/day {12.8 ft/day) compared to 7.2 x 10-7 (2.4 x 10-6 ft/day) estimated for the till matrix. However, it has been reported that molecular diffusion is an important retardation mechanism that reduces the concentration of contaminants along these fractures with distance from the source. Also, the fractures constitute a small volume of pore space; ·therefore, the quantity {or flux) of water flowing along the fractures is small. Disposal of drilling fluids in glaciofluvial sediments is not recommended. The study at the Winderl site is evidence of the adverse environmental impact such disposal can lead to. The impact of drilling fluid disposal in till is dependent upon the geologic setting. Migration of the drilling fluid constituents will occur along fractures in the till; widespread contamination could result if these contaminants intersect penneable lenses. A subsurface investigation is necessary at the disposal sites in till to identify these permeable lenses and to determine if any nearby aquifers exist

Judicial Recognition of Hospital Independent Duty of Care to Patients: Hannola v. City of Lakewood

This Note will examine the court\u27s rationale in Hannola v City of Lakewood and the previous leading Ohio case on emergency room care, Cooper v. Sisters of Charity. This Note will similarly examine control tests of employment, the concept of apparent authority and the series of cases on independent duty of care which have been decided in the eleven years between Cooper and Hannola. It is the conclusion of this Note that the Hannola decision is more consistent with the realities of employment and service in the health care industry, and that the public policy arguments presented by the court require significant modifications in decisional rationale for medical negligence cases

Parameterized Strings: Algorithms and Data Structures

A parameterized string (p-string) T = T[1] T[2]...T[n] is a sophisticated string of length n composed of symbols from a constant alphabet Sigma and a parameter alphabet pi. Given a pair of p-strings S and T, the parameterized pattern matching (p-match) problem is to verify whether the individual constant symbols match and whether there exists a bijection between the parameter symbols of S and T. If the two conditions are met, S is said to be a p-match of T. A significant breakthrough in the p-match area is the prev encoding, which is proven to identify a p-match between S and T if and only if prev(S) == prev(T). In order to utilize suffix data structures in terms of p-matching, we must account for the dynamic nature of the parameterized suffixes (p-suffixes) of T, namely prev(T[ i...n]) ∀ i, 1 ≤ i ≤ n.;In this work, we propose transformative approaches to the direct parameterized suffix sorting (p-suffix sorting) problem by generating and sorting lexicographically numeric fingerprints and arithmetic codes that correspond to individual p-suffixes. Our algorithm to p-suffix sort via fingerprints is the first theoretical linear time algorithm for p-suffix sorting for non-binary parameter alphabets, which assumes that each code is represented by a practical integer. We eliminate the key problems of fingerprints by introducing an algorithm that exploits the ordering of arithmetic codes to sort p-suffixes in linear time on average.;The longest previous factor (LPF) problem is defined for traditional strings exclusively from the constant alphabet Sigma. We generalize the LPF problem to the parameterized longest previous factor (pLPF) problem defined for p-strings. Subsequently, we present a linear time solution to construct the pLPF array. Given our pLPF algorithm, we show how to construct the pLCP (parameterized longest common prefix) array in linear time. Our algorithm is further exploited to construct the standard LPF and LCP arrays all in linear time.;We then study the structural string (s-string), a variant of the p-string that extends the p-string alphabets to include complementary parameters that correspond to one another. The s-string problem involves the new encoding schemes sencode and compl in order to identify a structural match (s-match). Current s-match solutions use a structural suffix tree (s-suffix tree) to study structural matches in RNA sequences. We introduce the suffix array, LCP, and LPF data structures for the s-string encoding schemes. Using our new data structures, we identify the first suffix array solution to the s-match problem. Our algorithms and data structures are shown to apply to s-strings and also p-strings and traditional strings