The Role of Crassostrea Virginica Hemocytes in Shell Formation: Ex Vivo Mineral Deposition by Cultured Hemocytes

Abstract of Technical Paper Presented at the 103rd Annual Meeting National Shellfisheries Association Baltimore, Maryland March 27–31, 201

Type I Polyketide Synthases: Methodology, Biocatalysis, and Evaluation of Substrate Promiscuity.

The gram-positive prokaryotes of the Streptomyces genus are prolific producers of secondary metabolites including a plethora of complex polyketide compounds. These natural products are constructed through decarboxylative Claisen condensations of simple malonic acids from primary metabolism by multidomain, modular enzymes called polyketide synthases (PKS) in a manner analogous to an industrial assembly line. A prominent example of one such pathway is the pikromycin (Pik) cluster from S. venezuelae ATCC 15439, which biosynthesizes a suite of 12- and 14-membered macrolide antibiotics. This pathway has been a workhorse in the Sherman lab for in vivo work, in vitro biochemistry, and more recently, biocatalysis and in depth structural analysis. This dissertation describes synthetic chemistry, in vitro biochemistry, and in vitro biocatalysis focused on the final two PKS modules from the Pik cluster, PikAIII and PikAIV. First, the native pentaketide from the Pik pathway was chemically synthesized and employed to optimize in vitro biochemistry/biocatalysis with PikAIII-TE and PikAIII/PikAIV, culminating in a biocatalytic platform for macrolide production in 13 linear steps. Next, the native hexaketide from the Pik pathway was synthesized from fermentation derived 10-deoxymethynolide and employed to optimize in vitro biochemistry of PikAIV and excised Pik thioesterase (TE) domain, revealing the ability to control the catalytic cycle of PikAIV and gain entry to 12- or 14-membered macrolactones with greater than 10:1 selectivity for either ring size. Finally, we simulated “combinatorial biosynthesis” in a controlled in vitro environment with PikAIII-TE and PikAIII/PikAIV to identify catalytic bottlenecks using unnatural pentaketide substrates that mimic engineering early in the pathway. Analyses of results generated to date indict the TE domain as the bottleneck in combinatorial biosynthesis, and a crucial target for protein engineering.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/111627/1/dougland_1.pd

A cross-validated three-dimensional model of an englacial and subglacial drainage system in a High-Arctic glacier

The project was supported by the Danish Geological Society and the Arctic Research and Technology Society.Recent speleological surveys of meltwater drainage systems in cold and polythermal glaciers have documented dynamic englacial and in some cases subglacial conduits formed by the ‘cut-and-closure’ mechanism. Investigations of the spatial distribution of such conduits often require a combination of different methods. Here, we studied the englacial drainage system in the cold glacier Longyearbreen, Svalbard by combining speleological exploration of a 478 m long meltwater conduit with a high-resolution ground penetrating radar (GPR) survey with two different centre-frequencies (25 and 100 MHz). The results yielded a 3-D documentation of the present englacial drainage system. The study shows that the overall form of englacial conduits can be detected from velocity−depth converted GPR data, and that the 3-D model can facilitate a method to pinpoint the reflections in a radargram corresponding with the englacial drainage system, although fine detail cannot be resolved. Visible reflections approximately parallel to the mapped englacial water drainage system likely result from sediment incorporated in the ice or from abandoned parts of the englacial drainage system.Publisher PDFPeer reviewe

Analysing Habitat Connectivity and Home Ranges of Bigmouth Buffalo and Channel Catfish Using a Large-Scale Acoustic Receiver Network

The determination if fish movement of potadromous species is impeded in a river system is often difficult, particularly when timing and extent of movements are unknown. Furthermore, evaluating river connectivity poses additional challenges. Here, we used large-scale, long-term fish movement to study and identify anthropogenic barriers to movements in the Lake Winnipeg basin including the Red, Winnipeg, and Assiniboine rivers. In the frame of the project, 80 Bigmouth Buffalo (Ictiobus cyprinellus) and 161 Channel Catfish (Ictalurus punctatus) were tagged with acoustic transmitters. Individual fish were detected with an acoustic telemetry network. Movements were subsequently analyzed using a continuous-time Markov model (CTMM). The study demonstrated large home ranges in the Lake Winnipeg basin and evidence of frequent transborder movements between Canada and the United States. The study also highlighted successful downstream fish passage at some barriers, whereas some barriers limited or completely blocked upstream movement. This biological knowledge on fish movements in the Lake Winnipeg basin highlights the need for fish passage solutions at different obstructions

Elemental Uptake in Relation to Root Characteristics of Tall Fescue

HiMag, an accession of tall fescue (Festuca arundinacea Schreb.), was selected for high magnesium (Mg) concentration in leaves to reduce grass tetany risk to ruminants. However, the mechanism for enhanced Mg uptake in HiMag leaves has not been determined. The objective was to investigate if increased Mg uptake in HiMag could be explained by differences in elemental distribution among plant parts, root characteristics, or organic acid concentrations compared to its parental cultivars, ‘‘Kentucky 31’’ (KY31) and ‘‘Missouri 96’’ (MO96). The study was conducted on a surface-irrigated calcareous Portneuf silt loam (coarse-silty, mixed, mesic, Durinodic Xeric Haplocalcid). Vegetation and soil cores of 7.6-cm diameter were sampled to a 45-cm soil depth in 15-cm increments. Mass and ash were determined for leaves, crowns, and roots. Leaf area, root length, root area, root length density, elemental concentration, and uptake [potassium (K), calcium (Ca), Mg, sodium (Na), and phosphorus (P)], and malate and citrate concentrations also were determined. Leaf Mg concentration was higher in HiMag than parental cultivars. HiMag generally did not differ in crown and root elemental concentrations from its parents. Risk of causing grass tetany, indicated by leaf K/(Ca+Mg), was lower in HiMag than KY31 and MO96 in both 1994 (P=0.03) and 1995 (P=0.01). Root length, area, and mass were not related to cation concentrations in the three tall fescue accessions, suggesting that HiMag may have an active uptake or transport mechanism for Mg

The Nuclear Ionized Gas in the Radio Galaxy M84 (NGC 4374)

We present optical images of the nucleus of the nearby radio galaxy M84 (NGC 4374 = 3C272.1) obtained with the Wide Field/Planetary Camera 2 (WFPC2) aboard the Hubble Space Telescope (HST). Our three images cover the H$\alpha$ + [N II] emission lines as well as the V and I continuum bands. Analysis of these images confirms that the H$\alpha$ + [N II] emission in the central 5'' (410 pc) is elongated along position angle (P.A.) \approx 72\arcdeg, which is roughly parallel to two nuclear dust lanes.Our high-resolution images reveal that the H$\alpha$ + [N II] emission has three components, namely a nuclear gas disk,an `ionization cone', and outer filaments. The nuclear disk of ionized gas has diameter $\approx 1'' = 82$ pc and major axis P.A. \approx 58\arcdeg \pm 6\arcdeg. On an angular scale of 0\farcs5, the major axis of this nuclear gas disk is consistent with that of the dust. However, the minor axis of the gas disk (P.A. \approx 148\arcdeg) is tilted with respect to that of the filamentary H$\alpha$ + [N II] emission at distances > 2'' from the nucleus; the minor axis of this larger scale gas is roughly aligned with the axis of the kpc-scale radio jets (P.A. \approx 170\arcdeg). The ionization cone (whose apex is offset by \approx 0\farcs3 south of the nucleus) extends 2'' from the nucleus along the axis of the southern radio jet. This feature is similar to the ionization cones seen in some Seyfert nuclei, which are also aligned with the radio axes.Comment: 11 pages plus 4 figure

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/3/anie202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/2/anie202004991_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/1/anie202004991.pd

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/2/ange202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/1/ange202004991.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/3/ange202004991_am.pd

Crustal and Upper-mantle Structure Beneath Ice-covered Regions in Antarctica from S-wave Receiver Functions and Implications for Heat Flow

S-wave receiver functions (SRFs) are used to investigate crustal and upper-mantle structure beneath several ice-covered areas of Antarctica. Moho S-to-P (Sp) arrivals are observed at ~6–8 s in SRF stacks for stations in the Gamburtsev Mountains (GAM) and Vostok Highlands (VHIG), ~5–6 s for stations in the Transantarctic Mountains (TAM) and the Wilkes Basin (WILK), and ~3–4 s for stations in the West Antarctic Rift System (WARS) and the Marie Byrd Land Dome (MBLD). A grid search is used to model the Moho Sp conversion time with Rayleigh wave phase velocities from 18 to 30 s period to estimate crustal thickness and mean crustal shear wave velocity. The Moho depths obtained are between 43 and 58 km for GAM, 36 and 47 km for VHIG, 39 and 46 km for WILK, 39 and 45 km for TAM, 19 and 29 km for WARS and 20 and 35 km for MBLD. SRF stacks for GAM, VHIG, WILK and TAM show little evidence of Sp arrivals coming from upper-mantle depths. SRF stacks for WARS and MBLD show Sp energy arriving from upper-mantle depths but arrival amplitudes do not rise above bootstrapped uncertainty bounds. The age and thickness of the crust is used as a heat flow proxy through comparison with other similar terrains where heat flow has been measured. Crustal structure in GAM, VHIG and WILK is similar to Precambrian terrains in other continents where heat flow ranges from ~41 to 58 mW m−2, suggesting that heat flow across those areas of East Antarctica is not elevated. For the WARS, we use the Cretaceous Newfoundland–Iberia rifted margins and the Mesozoic-Tertiary North Sea rift as tectonic analogues. The low-to-moderate heat flow reported for the Newfoundland–Iberia margins (40–65 mW m−2) and North Sea rift (60–85 mW m−2) suggest that heat flow across the WARS also may not be elevated. However, the possibility of high heat flow associated with localized Cenozoic extension or Cenozoic-recent magmatic activity in some parts of the WARS cannot be ruled out