Structural Analysis of the BamA-B complex in Acinetobacter baumannii

There are 2 types of bacteria, gram-negative and gram-positive bacteria. Gram-negative bacteria have both a plasma membrane and an outer membrane, while gram-positive only have a plasma membrane. The outer membranes of gram-negative bacteria have outer membrane proteins which are essential for the bacteria’s survival. Also located within the outer membrane is a multicomponent protein complex named the beta-barrel assembly machinery (BAM) complex. BAM is responsible for folding and inserting outer membrane proteins into the outer membrane. This protein complex serves an essential role but not much is understood about its function. In this study, two subunits of the BAM complex (BamA and BamB) in Acinetobacter baumannii were recombinantly expressed in E. coli and then purified, crystallized and analyzed using biophysical methods in order to gain a better understanding of the A. baumannii BAM complex structure. In efforts to prepare the BamAB complex, two milligrams of BamA and two milligrams of BamB were mixed in buffer. The sample was ran through a size-exclusion column (Superdex 200 Increase GL) and analyzed for complex formation. Results showed we were not able to obtain a properly assembled complex with the current protocol, however, we will be screening different detergents in hopes of finding one that will foster BamA-B complex formation. The information gained from solving this structure will further aid in understanding the mechanisms of this important protein

Saccharomyces Cerevisiae Cdc7 Homology in Drosophila Melanogaster

Saccharomyces cerevisiae Dbf4(Dumbbell former 4) and Cdc7(Cell Division Cycle 7) form a complex that phosphorylates Mcm2 (Minichromosome maintenance 2) to initiate DNA replication. Cdc7 is a target for cancer research because there is a Cdc7 ortholog in humans that is necessary for DNA replication and cell survival. Our goal is to characterise a putative Cdc7 homolog in Drosophila melanogaster (dCdc7). We have previously shown that expression of the known Drosophila Dbf4 ortholog, Chiffon, and dCdc7 can rescue yeast cells deficient in active Cdc7. Our hypothesis is that the dCdc7 is activated by Chiffon to phosphorylate MCM2. To test this hypothesis, we will determine if Chiffon interacts with dCdc7 and if Chiffon triggers kinase activity of dCdc7. To do this we purified dCdc7, a putative kinase dead substitution mutant of dCdc7, Chiffon, and Chiffon truncations that are predicted to contain the interacting domains. To produce these proteins we performed a combination of subcloning, SLIC(sequence and ligation independent cloning), and MultiBac Baculovirus techniques. SLIC is a recently developed method of cloning that uses single strand sequence complements instead of ligation to combine vector and insert. This allows us to clone any insert without regard to enzyme restriction sites within the insert’s DNA sequence. MultiBac is a system that allows us to infect insect cells with a Baculovirus to coexpress multiple proteins. We purified our proteins through affinity chromatography using various resins that bind to tags on the proteins. These proteins will then be used for protein binding pulldowns and kinase activity assays to determine if Chiffon and dCdc7 have a physical interaction, and if dCdc7 is a kinase. The results from the protein binding pulldown and kinase assay are pending. The results from these experiments will show us whether or not this protein is the Cdc7 ortholog in Drosophila

Regulation of DNA Synthesis: The Identification of New Drosophila melanogaster Cdc7 Regulatory Subunits

Cell division cycle 7 (Cdc7) is an enzyme required for the initiation of DNA replication. Cdc7 cannot act alone requiring the binding of its regulatory subunit Dbf4 to perform its enzymatic function. Previous studies show that Dbf4 and Cdc7 are well conserved across eukaryotic organisms. Humans and Xenopus have multiple Cdc7 regulatory subunits, and recent studies suggest that Drosophila melanogaster might as well. Human Dbf4 was discovered because of its similarity to yeast Dbf4. It is possible that finding additional Cdc7 regulatory subunits in D. melanogaster could reveal related proteins in humans. As cancer is a disease caused by improper cell cycling, furthering our understanding of Cdc7 and the cell cycle regulation could lead to advances in cancer treatment. This study seeks to identify possible Cdc7 regulatory subunits by screening for D. melanogaster proteins that directly interact with Cdc7. The first goal was to use a Yeast 2-Hybrid assay to repeat results that indicated an interaction between Cdc7 and Drosophila Dbf4, known as Chiffon. This allowed for testing media and the effectiveness of the assay. While not preformed yet, screening will be completed using a Yeast 2-Hybrid assay to determine interactions between Cdc7 and proteins from a D. melanogaster cDNA library. Further testing will remove false positives. Any remaining plasmids be sequenced and identified by the sequence comparison software, BLAST. Our study will test for D. melanogaster proteins that interact with Cdc7, but once these proteins are found further experimentation will be required to confirm interaction and function with Cdc7

Active Vibration Control Device

An active vibration control device for controlling vibration in a cantilevered member and a method for the same are disclosed. The device is comprised of a cantilevered member having a longitudinal axis comprising a sensor mounted near the free end of the member to measure motion of the member in a transverse direction and to produce a corresponding signal. A force generating assembly is mounted to the member near the free end to oppose the measured motion with a force thereby minimizing subsequent motion along the transverse axis caused by vibration

Use Of Honeycomb Seals In Stabilizing Two Centrifugal Compressors

LecturePg. 3-16The stabilizing effects achieved by replacing labyrinth seals with honeycomb seals are described. Although honeycomb seals have been used for some time in high pressure compressors and other turbomachinery applications, their stabilizing influence has only recently been recognized. The pioneering work of Childs et al. [1,2,3], in the theoretical analyses and accompanying experimental investigations have had a significant influence on advancing the use of honeycomb seal technology. A major beneficiary of the application of honeycomb seals has been the space shuttle turbo pumps. There are other major applications for honeycomb seals but, for the most part, they have remained in a narrow area encompassing the aerospace industry and the higher performance turbomachinery applications. The authors address how honeycomb seals were used to stabilize two process-type compressors. Two stability problems experienced on two different compressor configurations were solved using a common approach. These two cases should serve to enlighten users who might experience similar problems. It also sheds light on other sources of instability in turbo machinery, and some of the means available to counter their destabilizing effects. In both cases, the application of honeycomb seals was instrumental in enhancing the stability of each of the compressors

Effects of potential detoxifying agents on growth performance and deoxynivalenol (DON) urinary balance characteristics of nursery pigs fed DON-contaminated wheat

Citation: Frobose, H. L., Stephenson, E. W., Tokach, M. D., DeRouchey, J. M., Woodworth, J. C., Dritz, S. S., & Goodband, R. D. (2017). Effects of potential detoxifying agents on growth performance and deoxynivalenol (DON) urinary balance characteristics of nursery pigs fed DON-contaminated wheat. Journal of Animal Science, 95(1), 327-337. doi:10.2527/jas2016.0664Two experiments were conducted to evaluate potential detoxifying agents on growth of nursery pigs fed deoxynivalenol (DON)-contaminated diets. Naturally DON-contaminated wheat (6 mg/kg) was used to achieve desired DON levels. In a 21-d study, 238 pigs (13.4 +/- 1.8 kg BW) were used in a completely randomized design with a 2 x 2 + 1 factorial arrangement. Diets were: 1) Positive control (PC; < 0.5 mg/kg DON), 2) PC + 1.0% Product V (Nutriquest LLC, Mason City, IA), 3) Negative control (NC; 4.0 mg/kg DON), 4) NC + 1.0% Product V, and 5) NC + 1.0% sodium metabisulfite (SMB; Samirian Chemicals, Campbell, CA). There were 6 or 7 replicate pens/treatment and 7 pigs/pen. Analyzed DON was decreased by 92% when pelleted with SMB, but otherwise matched formulated levels. Overall, a DON x Product V interaction was observed for ADG (P < 0.05) with a tendency for an interaction for ADFI (P < 0.10). As anticipated, DON reduced (P < 0.001) ADG and ADFI, but the interaction was driven by even poorer growth when Product V was added to NC diets. Pigs fed NC diets had 10% poorer G: F (P < 0.001) than PC-fed pigs. Reductions in ADG due to DON were most distinct (50%) during the initial period. Adding SMB to NC diets improved (P < 0.01) ADG, ADFI, and G: F, and improved (P < 0.02) ADG and G: F compared to the PC diet. A urinary balance study was conducted using diets 3 to 5 from Exp. 1 to evaluate Product V and SMB on DON urinary metabolism. A 10 d adaptation was followed by a 7 d collection using 24 barrows in a randomized complete block design. Pigs fed NC + SMB diet had greater urinary DON output (P < 0.05) than pigs fed NC + Product V, with NC pigs intermediate. Daily DON excretion was lowest (P < 0.05) in the NC + SMB pigs. However, degradation of DON-sulfonate back to the parent DON molecule was observed as pigs fed NC + SMB excreted more DON than they consumed (164% of daily DON intake), greater (P < 0.001) than pigs fed the NC (59%) or NC + Product V (48%). Overall, Product V did not alleviate DON effects on growth nor did it reduce DON absorption and excretion. However, hydrothermally processing DON-contaminated diets with 1.0% SMB restored ADFI and improved G: F. Even so, the urinary balance experiment revealed that some of the converted DON-sulfonate can degrade back to DON under physiological conditions. While further research is needed to discern the stability of the DON-sulfonate, SMB appears promising to restore performance in pelleted DON-contaminated diets

The Extrasolar Planet epsilon Eridani b - Orbit and Mass

Hubble Space Telescope observations of the nearby (3.22 pc), K2 V star epsilon Eridani have been combined with ground-based astrometric and radial velocity data to determine the mass of its known companion. We model the astrometric and radial velocity measurements simultaneously to obtain the parallax, proper motion, perturbation period, perturbation inclination, and perturbation size. Because of the long period of the companion, \eps b, we extend our astrometric coverage to a total of 14.94 years (including the three year span of the \HST data) by including lower-precision ground-based astrometry from the Allegheny Multichannel Astrometric Photometer. Radial velocities now span 1980.8 -- 2006.3. We obtain a perturbation period, P = 6.85 +/- 0.03 yr, semi-major axis, alpha =1.88 +/- 0.20 mas, and inclination i = 30.1 +/- 3.8 degrees. This inclination is consistent with a previously measured dust disk inclination, suggesting coplanarity. Assuming a primary mass M_* = 0.83 M_{\sun}, we obtain a companion mass M = 1.55 +/- 0.24 M_{Jup}. Given the relatively young age of epsilon Eri (~800 Myr), this accurate exoplanet mass and orbit can usefully inform future direct imaging attempts. We predict the next periastron at 2007.3 with a total separation, rho = 0.3 arcsec at position angle, p.a. = -27 degrees. Orbit orientation and geometry dictate that epsilon Eri b will appear brightest in reflected light very nearly at periastron. Radial velocities spanning over 25 years indicate an acceleration consistent with a Jupiter-mass object with a period in excess of 50 years, possibly responsible for one feature of the dust morphology, the inner cavity

Patterns of Natural and Human-Caused Mortality Factors of a Rare Forest Carnivore, the Fisher (Pekania pennanti) in California.

Wildlife populations of conservation concern are limited in distribution, population size and persistence by various factors, including mortality. The fisher (Pekania pennanti), a North American mid-sized carnivore whose range in the western Pacific United States has retracted considerably in the past century, was proposed for threatened status protection in late 2014 under the United States Endangered Species Act by the United States Fish and Wildlife Service in its West Coast Distinct Population Segment. We investigated mortality in 167 fishers from two genetically and geographically distinct sub-populations in California within this West Coast Distinct Population Segment using a combination of gross necropsy, histology, toxicology and molecular methods. Overall, predation (70%), natural disease (16%), toxicant poisoning (10%) and, less commonly, vehicular strike (2%) and other anthropogenic causes (2%) were causes of mortality observed. We documented both an increase in mortality to (57% increase) and exposure (6%) from pesticides in fishers in just the past three years, highlighting further that toxicants from marijuana cultivation still pose a threat. Additionally, exposure to multiple rodenticides significantly increased the likelihood of mortality from rodenticide poisoning. Poisoning was significantly more common in male than female fishers and was 7 times more likely than disease to kill males. Based on necropsy findings, suspected causes of mortality based on field evidence alone tended to underestimate the frequency of disease-related mortalities. This study is the first comprehensive investigation of mortality causes of fishers and provides essential information to assist in the conservation of this species

Some New Results on Complex-Temperature Singularities in Potts Models on the Square Lattice

We report some new results on the complex-temperature (CT) singularities of $q$-state Potts models on the square lattice. We concentrate on the problematic region $Re(a) < 0$ (where $a=e^K$) in which CT zeros of the partition function are sensitive to finite lattice artifacts. From analyses of low-temperature series expansions for $3 \le q \le 8$, we establish the existence, in this region, of complex-conjugate CT singularities at which the magnetization and susceptibility diverge. From calculations of zeros of the partition function, we obtain evidence consistent with the inference that these singularities occur at endpoints $a_e, \ a_e^*$ of arcs protruding into the (complex-temperature extension of the) FM phase. Exponents for these singularities are determined; e.g., for $q=3$, we find $\beta_e=-0.125(1)$, consistent with $\beta_e=-1/8$. By duality, these results also imply associated arcs extending to the (CT extension of the) symmetric PM phase. Analytic expressions are suggested for the positions of some of these singularities; e.g., for $q=5$, our finding is consistent with the exact value $a_e,a_e^*=2(-1 \mp i)$. Further discussions of complex-temperature phase diagrams are given.Comment: 26 pages, latex, with eight epsf figure