Cell Biology & Molecular Genetics
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Item Carbohydrase Systems of Saccharophagus degradans Degrading Marine Complex Polysaccharides(MDPI, 2011-04-21) Hutcheson, Steven W.; Zhang, Haitao; Suvorov, MaximSaccharophagus degradans 2–40 is a γ-subgroup proteobacterium capable of using many of the complex polysaccharides found in the marine environment for growth. To utilize these complex polysaccharides, this bacterium produces a plethora of carbohydrases dedicated to the processing of a carbohydrate class. Aiding in the identification of the contributing genes and enzymes is the known genome sequence for this bacterium. This review catalogs the genes and enzymes of the S. degradans genome that are likely to function in the systems for the utilization of agar, alginate, α- and β-glucans, chitin, mannans, pectins, and xylans and discusses the cell biology and genetics of each system as it functions to transfer carbon back to the bacterium.Item IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts(MDPI, 2014-03-24) Hoover, Lisa I.; Fredericksen, Brenda L.Although dermal fibroblasts are one of the first cell types exposed to West Nile virus (WNV) during a blood meal by an infected mosquito, little is known about WNV replication within this cell type. Here, we demonstrate that neuroinvasive, WNV-New York (WNV-NY), and nonneuroinvasive, WNV-Australia (WNV-AUS60) strains are able to infect and replicate in primary human dermal fibroblasts (HDFs). However, WNV-AUS60 replication and spread within HDFs was reduced compared to that of WNV-NY due to an interferon (IFN)-independent reduction in viral infectivity early in infection. Additionally, replication of both strains was constrained late in infection by an IFN-β-dependent reduction in particle infectivity. Overall, our data indicates that human dermal fibroblasts are capable of supporting WNV replication; however, the low infectivity of particles produced from HDFs late in infection suggests that this cell type likely plays a limited role as a viral reservoir in vivo.Item Generation of West Nile Virus Infectious Clones Containing Amino Acid Insertions Between Capsid and Capsid Anchor(MDPI, 2014-04-09) Vandergaast, Rianna; Hoover, Lisa I.; Zheng, Kang; Fredericksen, Brenda L.West Nile virus (WNV) is a positive-sense RNA arbovirus responsible for recent outbreaks of severe neurological disease within the US and Europe. Large-scale analyses of antiviral compounds that inhibit virus replication have been limited due to the lack of an adequate WN reporter virus. Previous attempts to insert a reporter into the 3’ untranslated region of WNV generated unstable viruses, suggesting that this region does not accommodate additional nucleotides. Here, we engineered two WNV infectious clones containing insertions at the Capsid (C)/Capsid Anchor (CA) junction of the viral polyprotein. Recombinant viruses containing a TAT(1-67) or Gaussia Luciferase (GLuc) gene at this location were successfully recovered. However, rapid loss of most, if not all, of the reporter sequence occurred for both viruses, indicating that the reporter viruses were not stable. While the GLuc viruses predominantly reverted back to wild-type WNV length, the TAT viruses retained up to 75 additional nucleotides of the reporter sequence. These additional nucleotides were stable over at least five passages and did not significantly alter WNV fitness. Thus, the C/CA junction of WNV can tolerate additional nucleotides, though insertions are subject to certain constraints.Item The Role of Potassium Channels in Arabidopsis thaliana Long Distance Electrical Signalling: AKT2 Modulates Tissue Excitability While GORK Shapes Action Potentials(MDPI, 2018-03-21) Cuin, Tracey Ann; Dreyer, Ingo; Michard, ErwanFast responses to an external threat depend on the rapid transmission of signals through a plant. Action potentials (APs) are proposed as such signals. Plant APs share similarities with their animal counterparts; they are proposed to depend on the activity of voltage-gated ion channels. Nonetheless, despite their demonstrated role in (a)biotic stress responses, the identities of the associated voltage-gated channels and transporters remain undefined in higher plants. By demonstrating the role of two potassium-selective channels in Arabidopsis thaliana in AP generation and shaping, we show that the plant AP does depend on similar Kv-like transport systems to those of the animal signal. We demonstrate that the outward-rectifying potassium-selective channel GORK limits the AP amplitude and duration, while the weakly-rectifying channel AKT2 affects membrane excitability. By computational modelling of plant APs, we reveal that the GORK activity not only determines the length of an AP but also the steepness of its rise and the maximal amplitude. Thus, outward-rectifying potassium channels contribute to both the repolarisation phase and the initial depolarisation phase of the signal. Additionally, from modelling considerations we provide indications that plant APs might be accompanied by potassium waves, which prime the excitability of the green cable.Item Neisseria gonorrhoeae Aggregation Reduces Its Ceftriaxone Susceptibility(MDPI, 2018-06-15) Wang, Liang-Chun; Litwin, Madeline; Sahiholnasab, Zahraossadat; Song, Wenxia; Stein, Daniel C.Antibiotic resistance in Neisseria gonorrhoeae (GC) has become an emerging threat worldwide and heightens the need for monitoring treatment failures. N. gonorrhoeae, a gram-negative bacterium responsible for gonorrhea, infects humans exclusively and can form aggregates during infection. While minimal inhibitory concentration (MIC) tests are often used for determining antibiotic resistance development and treatment, the knowledge of the true MIC in individual patients and how it relates to this laboratory measure is not known. We examined the effect of aggregation on GC antibiotic susceptibility and the relationship between bacterial aggregate size and their antibiotic susceptibility. Aggregated GC have a higher survival rate when treated with ceftriaxone than non-aggregated GC, with bacteria in the core of the aggregates surviving the treatment. GC lacking opacity-associated protein or pili, or expressing a truncated lipooligosaccharide, three surface molecules that mediate GC-GC interactions, reduce both aggregation and ceftriaxone survival. This study demonstrates that the aggregation of N. gonorrhoeae can reduce the susceptibility to antibiotics, and suggests that antibiotic utilization can select for GC surface molecules that promote aggregation which in turn drive pathogen evolution. Inhibiting aggregation may be a potential way of increasing the efficacy of ceftriaxone treatment, consequently reducing treatment failure.Item The Expanding Riboverse(MDPI, 2019-10-05) Sulima, Sergey O.; Dinman, Jonathan D.Subverting the conventional concept of “the” ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality than has traditionally been thought possible. A diverse array of researchers is collectively illuminating a universe of heterogeneous and adaptable ribosomes harboring differences in composition and regulatory capacity: These differences enable specialization. The expanding universe of ribosomes not only comprises an incredible richness in ribosomal specialization between species, but also within the same tissues and even cells. In this review, we discuss ribosomal heterogeneity and speculate how the emerging understanding of the ribosomal repertoire is impacting the biological sciences today. Targeting pathogen-specific and pathological “diseased” ribosomes promises to provide new treatment options for patients, and potential applications for “designer ribosomes” are within reach. Our deepening understanding of and ability to manipulate the ribosome are establishing both the technological and theoretical foundations for major advances for the 21st century and beyond.Item Collaborative learning in an undergraduate life sciences living-learning program: case studies at multiple planes of analysis.(2017) Jardine, Hannah; Levin, Daniel; Quimby, B. Bryn; Cooke, ToddThe authors report on a living-learning program (LLP) designed to transform life sciences education. One goal of the LLP is to engage students in collaborative learning. Little research describes interactions and experiences within an LLP that encourage collaborative learning. This qualitative ethnographic study explores the following questions: What are some of the ways in which collaborative learning occurs in an LLP? and What factors influence how, when, and to what extent collaborative learning occurs in an LLP? The authors aim to identify ways to promote collaborative learning in an LLP and provide insight for others wishing to construct LLPs with similar goals.Item Examining the impact of student expectations on undergraduate biology education reform.(2011) Hall, Kristi; Watkins, Jessica; Coffey, Janet; Cooke, Todd; Redish, EdwardThe past 10-15 years have seen numerous calls for curricular reform in undergraduate biology education, most of which focus on changes to curriculum or pedagogy. Data collected from students in a large introductory undergraduate biology course indicate that student expectations about the nature of the knowledge they were learning influence how they interacted with reform efforts in that class. Given that student expectations influence the ways in which they participate in course activities, this paper (the first in a series that looks at student expectations in biology) argues that curriculum reform initiatives should consider student expectations in order to increase the chance for effective implementation.Item A sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages(Springer Nature, 2005-05-24) Gonçalves, Ricardo; Vieira, Etel R; Melo, Maria N; Gollob, Kenneth J; Mosser, David M; Tafuri, Wagner LThe Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay. To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry. Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) β2 integrin. Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.Item SNPs3D: Candidate gene and SNP selection for association studies(Springer Nature, 2006-03-22) Yue, Peng; Melamud, Eugene; Moult, JohnThe relationship between disease susceptibility and genetic variation is complex, and many different types of data are relevant. We describe a web resource and database that provides and integrates as much information as possible on disease/gene relationships at the molecular level. The resource http://www.SNPs3D.org has three primary modules. One module identifies which genes are candidates for involvement in a specified disease. A second module provides information about the relationships between sets of candidate genes. The third module analyzes the likely impact of non-synonymous SNPs on protein function. Disease/candidate gene relationships and gene-gene relationships are derived from the literature using simple but effective text profiling. SNP/protein function relationships are derived by two methods, one using principles of protein structure and stability, the other based on sequence conservation. Entries for each gene include a number of links to other data, such as expression profiles, pathway context, mouse knockout information and papers. Gene-gene interactions are presented in an interactive graphical interface, providing rapid access to the underlying information, as well as convenient navigation through the network. Use of the resource is illustrated with aspects of the inflammatory response and hypertension. The combination of SNP impact analysis, a knowledge based network of gene relationships and candidate genes, and access to a wide range of data and literature allow a user to quickly assimilate available information, and so develop models of gene-pathway-disease interaction.