From Behavior to Genes, and Back Again.

The aim of this thesis was to use forward and reverse genetics to explore the contribution of unknown and known genes to the touch-evoked escape behaviors belonging to zebrafish (Danio rerio). A forward genetic screen identified two mutants that displayed abnormal touch-evoked escape behaviors. The first mutant touchdown (tdo) lacked sensitivity to light touch, but responded to noxious stimuli. tdo mutants were shown to result from mutations in the gene encoding for TRPM7, an ion channel with an attached kinase. A combination of electrophysiological and molecular techniques revealed that ion channel function was sufficient to restore sensitivity to light touch within excitable mechanosensitive neurons. Therefore TRPM7 is a candidate for a vertebrate mechanoreceptor responsible for sensitivity to light touch. The second mutant identified from our forward genetic screen non-active (nav) initiated escape contractions, but failed to swim in response to touch. nav mutants were shown to result from mutations in the gene encoding NaV1.6 that abolished channel activity. Electrophysiological recordings revealed that NaV1.6 is required to turn on a locomotor network capable of generating rudimentary swimming. Furthermore the requirement of NaV1.6 to turn this network might be its contribution of a persistent sodium current which is known to facilitate repetitive firing in other neurons. Finally the cloning and characterization of the ATP-gated P2X receptor subunits P2X1 through P2X5.2 from zebrafish demonstrated that two subunits (P2X1 and P2X5.1) form functional homomeric receptors in contrast to previous reports. The use of reverse genetics (antisense knockdown) allowed the rejection of the hypothesis that signaling through P2X receptors is essential for myogenesis.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60889/1/selow_1.pd

Implementing Primordial Binaries in Simulations of Star Cluster Formation with a Hybrid MHD and Direct N-Body Method

The fraction of stars in binary systems within star clusters is important for their evolution, but what proportion of binaries form by dynamical processes after initial stellar accretion remains unknown. In previous work, we showed that dynamical interactions alone produced too few low-mass binaries compared to observations. We therefore implement an initial population of binaries in the coupled MHD and direct N-body star cluster formation code Torch. We compare simulations with, and without, initial binary populations and follow the dynamical evolution of the binary population in both sets of simulations, finding that both dynamical formation and destruction of binaries take place. Even in the first few million years of star formation, we find that an initial population of binaries is needed at all masses to reproduce observed binary fractions for binaries with mass ratios above the $q \geq 0.1$ detection limit. Our simulations also indicate that dynamical interactions in the presence of gas during cluster formation modify the initial distributions towards binaries with smaller primary masses, larger mass ratios, smaller semi-major axes and larger eccentricities. Systems formed dynamically do not have the same properties as the initial systems, and systems formed dynamically in the presence of an initial population of binaries differ from those formed in simulations with single stars only. Dynamical interactions during the earliest stages of star cluster formation are important for determining the properties of binary star systems.Comment: 15 pages, 14 figures, submitted to MNRAS and edited to address positive referee's repor

The Grizzly, January 28, 2016

Student Raises Funds to Prevent Suicide • Tragedy Prompts Main Street Changes • Ursinus Introduces Open Access Publishing System • Ursinus Welcomes Alumni as Entrepreneur-in-Residence • International Perspective: Differences in Friendships Across Cultures • Rare Bird Exhibit Migrates to Berman • Coasting Through Costa Rica • Opinions: Everyone\u27s an Activist on Facebook; Letter from an Alumnus: Guns; Film Review: Star Wars: The Force Awakens Rates 7 / 10 • Women\u27s Indoor Track and Field Ready to Run • Vaulting Into Actionhttps://digitalcommons.ursinus.edu/grizzlynews/1680/thumbnail.jp

Na V 1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non-active ( nav mi89 and nav mi130 ). nav embryos displayed diminished spontaneous and touch-evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na V 1.6a ( scn8aa ) as a potential candidate for nav . Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na V 1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild-type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav . In-vivo electrophysiological analysis of the touch-evoked escape circuit indicated that voltage-dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na V 1.6a was required for touch-induced activation of the swim locomotor network. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508–522, 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75774/1/20791_ftp.pd

A comparison of economy and sagittal plane trunk movements among back-, back/front- and head-loading.

It has been suggested that freedom of movement in the trunk could influence load carriage economy. This study aimed to compare the economy and sagittal plane trunk movements associated with three load carriage methods that constrain posture differently. Eighteen females walked at 3 km.h-1 with loads of 0, 3, 6, 9, 12, 15 and 20 kg carried on the back, back/front and head. Load carriage economy was assessed using the Extra Load Index (ELI). Change in sagittal plane trunk forward lean and trunk angle excursion from unloaded to loaded walking were assessed. Results show no difference in economy between methods (p = 0.483), despite differences in the change in trunk forward lean (p = 0.001) and trunk angle excursion (p = 0.021) from unloaded to loaded walking. We conclude that economy is not different among the three methods of load carriage, despite significant differences in sagittal plane trunk movements

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

X-Rays from Superbubbles in the Large Magellanic Cloud. V. The H II Complex N11

The large H II complex N11 in the Large Magellanic Cloud contains OB associations at several different stages in their life histories. We have obtained ROSAT PSPC and HRI X-ray observations, Curtis Schmidt CCD images, echelle spectra in H-alpha and [N II] lines, and IUE interstellar absorption line observations of this region. The central bubble of N11 has an X-ray luminosity a factor of only 3-7 brighter than predicted for an energy-conserving superbubble, making this the first detection of X-ray emission from a superbubble without a strong X-ray excess. The region N11B contains an extremely young OB association analogous to the central association of the Carina nebula, apparently still embedded in its natal molecular cloud. We find that N11B emits diffuse X-ray emission, probably powered by stellar winds. Finally, we compare the tight cluster HD32228 in N11 to R136 in 30 Dor. The latter is a strong X-ray source, while the former is not detected, showing that strong X-ray emission from compact objects is not a universal property of such tight clusters.Comment: submitted to ApJ 1 April 1997, uses aasms4.sty, 20 pages, 10 figures (figure 3 is color; figures 1a and 4 are gifs; original postscript available from http://www.mpia-hd.mpg.de/MPIA/Projects/THEORY/maclow/papers/n11/n11.htm

Loss of Myotubularin Function Results in T-Tubule Disorganization in Zebrafish and Human Myotubular Myopathy

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation–contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis

Towards a Synthetic Chloroplast

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London