Requirements for Catalysis in Cre Recombinase

Cre recombinase, a member of the tyrosine recombinase (YR) family of site-specific recombinases catalyzes DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). In this dissertation, the requirements for YR catalysis and the relationship between the YRs and the TopIBs are explored. I have analyzed the in vivo and in vitro recombination activities of all possible substitutions of the seven active site residues in Cre recombinase. To facilitate the interpretation mutant activities, I also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that allows for a comparison with similar structures from the related TopIBs. The results demonstrate that active site residues shared by the TopIBs are most sensitive to substitution. Two of the conserved active site residues in YRs have no equivalent in TopIBs. I have concluded that Glu176 and His289 in Cre evolved to have functional roles in site-specific recombination, that are unnecessary for relaxation by TopIB. His289 is not essential for cleavage of DNA, but accelerates water mediated hydrolysis of the 3\u27-phosphotyrosine covalent intermediate. Glu176 serves a structural role in facilitating the activation of Cre for cleavage by helping position the general acid, K201 in the active site. These residues may compensate for activity lost during the evolution of allosteric regulation required for recombination. Examination of two regions involved in protein:protein interactions in the Cre-DNA tetramer complex has led to the characterization of two modes of allosteric regulation. The first involves a C-terminal helix (hN), which binds in the pocket of a neighboring Cre monomer to serve as a regulatory switch by acting as a tether to position the tyrosine nucleophile (Y324). The second regulatory module involves the mobile β2-β3 hairpin carrying the general acid, K201. This hairpin must form synapsis dependent contacts to efficiently position K201 near O5\u27 of the scissile phosphate. Cre has evolved to modulate the positions of critical residues for catalysis, which is an effective and highly sensitive mechanism of regulation of catalysis not shared by TopIBs

Neural Compensations After Lesion of the Cerebral Cortex

Functional improvement after cortical injury can be stimulated by various factors including experience, psychomotor stimulants, gonadal hormones, and neurotrophic factors. The, timing of the administration of these factors may be critical, however. For example, factors such as gonadal hormones, nerve growth factor, or psychomotor stimulants may act to either enhance or retard recovery, depending upon the timing of administration. Nicotine, for instance, stimulates recovery if given after an injury but is without neuroprotective effect and may actually retard recovery if it is given only preinjury. A related timing problem concerns the interaction of different treatments. For example, behavioral therapies may act, in part, via their action in stimulating the endogenous production of trophic factors. Thus, combining behavioral therapies with pharmacological administration of compounds to increase the availability of trophic factors enhances functional outcome. Finally, anatomical evidence suggests that the mechanism of action of many treatments is through changes in dendritic arborization, which presumably reflects changes in synaptic organization. Factors that enhance dendritic change stimulate functional compensation, whereas factors that retard or block dendritic change block or retard compensation

Plasticity in the prefrontal cortex of adult rats

Sherpa Romeo green journal, open accessWe review the plastic changes of the prefrontal cortex of the rat in response to a wide range of experiences including sensory and motor experience, gonadal hormones, psychoactive drugs, learning tasks, stress, social experience, metaplastic experiences, and brain injury. Our focus is on synaptic changes (dendritic morphology and spine density) in pyramidal neurons and the relationship to behavioral changes. The most general conclusion we can reach is that the prefrontal cortex is extremely plastic and that the medial and orbital prefrontal regions frequently respond very differently to the same experience in the same brain and the rules that govern prefrontal plasticity appear to differ for those of other cortical regions.Ye

Prenatal enrichment and recovery from perinatal cortical damage: effects of maternal complex housing

Open access journalBirth is a particularly vulnerable time for acquiring brain injury. Unfortunately, very few treatments are available for those affected. Here we explore the effectiveness of prenatal intervention in an animal model of early brain damage. We used a complex housing paradigm as a form of prenatal enrichment. Six nulliparous dams and one male rat were placed in complex housing (condomom group) for 12 h per day until the dams’ delivered their pups. At parturition the dams were left in their home (standard) cages with their pups. Four dams were housed in standard cages (cagemom group) throughout pregnancy and with their pups until weaning. At postnatal day 3 (P3) infants of both groups received frontal cortex removals or sham surgery. Behavioral testing began on P60 and included the Morris water task and a skilled reaching task. Brains were processed for Golgi analyses. Complex housing of the mother had a significant effect on the behavior of their pups. Control animals from condomom group outperformed those of the cagemom group in the water task. Condomom animals with lesions performed better than their cagemom cohorts in both the water task and in skilled reaching. Codomom animals showed an increase in cortical thickness at anterior planes and thalamic area at both anterior and posterior regions. Golgi analyses revealed an increase in spine density. These results suggest that prenatal enrichment alters brain organization in manner that is prophylactic for perinatal brain injury. This result could have significant implications for the prenatal management of infants expected to be at risk for difficult birth.Ye

A Comparative Astrochemical Study Of The High-Mass Protostellar Objects NGC 7538 IRS 9 and IRS 1

We report the results of a spectroscopic study of the high-mass protostellar object NGC 7538 IRS 9 and compare our observations to published data on the nearby object NGC 7538 IRS 1. Both objects originated in the same molecular cloud and appear to be at different points in their evolutionary histo- ries, offering an unusual opportunity to study the temporal evolution of envelope chemistry in objects sharing a presumably identical starting composition. Observations were made with the Texas Echelon Cross Echelle Spectrograph (TEXES), a sensitive, high spectral resolution (R = {\lambda}/{\Delta}{\lambda} \simeq 100,000) mid-infrared grating spectrometer. Forty-six individual lines in vibrational modes of the molecules C2H2, CH4, HCN, NH3 and CO were detected, including two isotopologues (13CO, 12C18O) and one combination mode ({\nu}4 + {\nu}5 C2H2). Fitting synthetic spectra to the data yielded the Doppler shift, excitation temperature, Doppler b parameter, column density and covering factor for each molecule observed; we also computed column density upper limits for lines and species not detected, such as HNCO and OCS. We find differences among spectra of the two objects likely attributable to their differing radiation and thermal environments. Temperatures and column densities for the two objects are generally consistent, while the larger line widths toward IRS 9 result in less saturated lines than those toward IRS 1. Finally, we compute an upper limit on the size of the continuum-emitting region (\sim2000 AU) and use this constraint and our spectroscopy results to construct a schematic model of IRS 9.Comment: 23 pages, 15 figures, 6 tables; accepted for publication in Ap

Deconvolution of Images from BLAST 2005: Insight into the K3-50 and IC 5146 Star-Forming Regions

We present an implementation of the iterative flux-conserving Lucy-Richardson (L-R) deconvolution method of image restoration for maps produced by the Balloon-borne Large Aperture Submillimeter Telescope (BLAST). We have analyzed its performance and convergence extensively through simulations and cross-correlations of the deconvolved images with available highresolution maps. We present new science results from two BLAST surveys, in the Galactic regions K3-50 and IC 5146, further demonstrating the benefits of performing this deconvolution. We have resolved three clumps within a radius of 4.'5 inside the star-forming molecular cloud containing K3-50. Combining the well-resolved dust emission map with available multi-wavelength data, we have constrained the Spectral Energy Distributions (SEDs) of five clumps to obtain masses (M), bolometric luminosities (L), and dust temperatures (T). The L-M diagram has been used as a diagnostic tool to estimate the evolutionary stages of the clumps. There are close relationships between dust continuum emission and both 21-cm radio continuum and 12CO molecular line emission. The restored extended large scale structures in the Northern Streamer of IC 5146 have a strong spatial correlation with both SCUBA and high resolution extinction images. A dust temperature of 12 K has been obtained for the central filament. We report physical properties of ten compact sources, including six associated protostars, by fitting SEDs to multi-wavelength data. All of these compact sources are still quite cold (typical temperature below ~ 16 K) and are above the critical Bonner-Ebert mass. They have associated low-power Young Stellar Objects (YSOs). Further evidence for starless clumps has also been found in the IC 5146 region.Comment: 13 pages, 12 Figures, 3 Table

An inclusive Research and Education Community (iREC) model to facilitate undergraduate science education reform

Funding: This work was supported by Howard Hughes Medical Institute grants to DIH is GT12052 and MJG is GT15338.Over the last two decades, there have been numerous initiatives to improve undergraduate student outcomes in STEM. One model for scalable reform is the inclusive Research Education Community (iREC). In an iREC, STEM faculty from colleges and universities across the nation are supported to adopt and sustainably implement course-based research – a form of science pedagogy that enhances student learning and persistence in science. In this study, we used pathway modeling to develop a qualitative description that explicates the HHMI Science Education Alliance (SEA) iREC as a model for facilitating the successful adoption and continued advancement of new curricular content and pedagogy. In particular, outcomes that faculty realize through their participation in the SEA iREC were identified, organized by time, and functionally linked. The resulting pathway model was then revised and refined based on several rounds of feedback from over 100 faculty members in the SEA iREC who participated in the study. Our results show that in an iREC, STEM faculty organized as a long-standing community of practice leverage one another, outside expertise, and data to adopt, implement, and iteratively advance their pedagogy. The opportunity to collaborate in this manner and, additionally, to be recognized for pedagogical contributions sustainably engages STEM faculty in the advancement of their pedagogy. Here, we present a detailed pathway model of SEA that, together with underpinning features of an iREC identified in this study, offers a framework to facilitate transformations in undergraduate science education.Peer reviewe

Riverine concentrations and export of dissolved silicon, and potential controls on nutrient stoichiometry, across the land–ocean continuum in Great Britain

Silicon (Si) is an essential nutrient element in freshwater and marine ecosystems, and its abundance relative to macro-nutrients (N, P) can impact phytoplankton communities in eutrophic rivers and estuaries. This study is the first national assessment examining (i) the primary sources (geological, biological, landcover) and controls (geomorphological, precipitation) on the transport of terrestrial dissolved silicon across Great Britain to the ocean, and (ii) the current extent and nature of its interactions with macro-nutrients in these catchments in relation to its potential impacts on phytoplankton community structure. It uses results from a year-long survey of 41 rivers along with historical data. Highest concentrations of dissolved Si (4–5.5 mg L-1) were found in rivers of the chalk- and sedimentary sandstone-based catchments of southern Great Britain and the hard sandstone catchments of Scotland. Catchment yield rates for dissolved Si varied between 0.2 and 2.6 t km−2 yr−1, with highest yields found in catchments with higher precipitation and runoff. Analysis of river N:P and dissolved Si:N ratios suggested that the sampled rivers were typically N enriched, and P limited with respect to dissolved Si. Molar dissolved Si:N ratios < 1, an indicator of river eutrophication, were associated with total nitrogen concentrations exceeding 1.8 mg L-1 or greater. The Indicator of Coastal Eutrophication index was used to assess the potential role of dissolved Si in the eutrophication of coastal waters. Negative values indicating limited eutrophication potential to non-siliceous algae were generally found, although some rivers had annual Indicator of Coastal Eutrophication index values exceeding 0, with values as high as 35 kg C km−2 day−1. In many eutrophic rivers, high dissolved Si concentrations derived from catchment lithology, kept the Indicator of Coastal Eutrophication index values below zero. Results have demonstrated that high N and P export have likely shifted most Great Britain rivers and coastal waters beyond the stoichiometric range where diatoms dominate production and into one where non-siliceous algae maybe increasingly present. Thus, future assessments of macro-nutrient management schemes, such as those involving wetlands should include dissolved Si routinely due to its stoichiometric importance

Dissolved inorganic carbon export from rivers of Great Britain: Spatial distribution and potential catchment-scale controls

Dissolved inorganic carbon (DIC) fluxes from the land to ocean have been quantified for many rivers globally. However, CO2 fluxes to the atmosphere from inland waters are quantitatively significant components of the global carbon cycle that are currently poorly constrained. Understanding, the relative contributions of natural and human-impacted processes on the DIC cycle within catchments may provide a basis for developing improved management strategies to mitigate free CO2 concentrations in rivers and subsequent evasion to the atmosphere. Here, a large, internally consistent dataset collected from 41 catchments across Great Britain (GB), accounting for ∼36% of land area (∼83,997 km2) and representative of national land cover, was used to investigate catchment controls on riverine dissolved inorganic carbon (DIC), bicarbonate (HCO3−) and free CO2 concentrations, fluxes to the coastal sea and annual yields per unit area of catchment. Estimated DIC flux to sea for the survey catchments was 647 kt DIC yr−1 which represented 69% of the total dissolved carbon flux from these catchments. Generally, those catchments with large proportions of carbonate and sedimentary sandstone were found to deliver greater DIC and HCO3− to the ocean. The calculated mean free CO2 yield for survey catchments (i.e. potential CO2 emission to the atmosphere) was 0.56 t C km−2 yr−1. Regression models demonstrated that whilst river DIC (R2 = 0.77) and HCO3− (R2 = 0.77) concentrations are largely explained by the geology of the landmass, along with a negative correlation to annual precipitation, free CO2 concentrations were strongly linked to catchment macronutrient status. Overall, DIC dominates dissolved C inputs to coastal waters, meaning that estuarine carbon dynamics are sensitive to underlying geology and therefore are likely to be reasonably constant. In contrast, potential losses of carbon to the atmosphere via dissolved CO2, which likely constitute a significant fraction of net terrestrial ecosystem production and hence the national carbon budget, may be amenable to greater direct management via altering patterns of land use

Sources, composition, and export of particulate organic matter across British estuaries

Estuaries receive and process a large amount of particulate organic carbon (POC) prior to its export into coastal waters. Studying the origin of this POC is key to understanding the fate of POC and the role of estuaries in the global carbon cycle. Here, we evaluated the concentrations of POC, as well as particulate organic nitrogen (PON), and used stable carbon and nitrogen isotopes to assess their sources across 13 contrasting British estuaries during five different sampling campaigns over 1 year. We found a high variability in POC and PON concentrations across the salinity gradient, reflecting inputs, and losses of organic material within the estuaries. Catchment land cover appeared to influence the contribution of POC to the total organic carbon flux from the estuary to coastal waters, with POC contributions >36% in estuaries draining catchments with a high percentage of urban/suburban land, and <11% in estuaries draining catchments with a high peatland cover. There was no seasonal pattern in the isotopic composition of POC and PON, suggesting similar sources for each estuary over time. Carbon isotopic ratios were depleted (−26.7 ± 0.42‰, average ± sd) at the lowest salinity waters, indicating mainly terrigenous POC (TPOC). Applying a two-source mixing model, we observed high variability in the contribution of TPOC at the highest salinity waters between estuaries, with a median value of 57%. Our results indicate a large transport of terrigenous organic carbon into coastal waters, where it may be buried, remineralized, or transported offshore