51 research outputs found
"Marker of Self" CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors
Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress âMarker of Selfâ CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show âhCD47-Lentiâ display properly oriented human-CD47 for interactions with the macrophage's inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rgâ/â (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known âSelfâ signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas
Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium "<em>Chlorochromatium aggregatum</em>"
BACKGROUND: âChlorochromatium aggregatumâ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. âChlorochromatium aggregatumâ is a motile, barrel-shaped aggregate formed from a single cell of âCandidatus Symbiobacter mobilisâ, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium. RESULTS: We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, âCa. S. mobilisâ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of âCa. S. mobilisâ on Chl. chlorochromatii is described. CONCLUSIONS: Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, âCa. S. mobilisâ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships
Genomic diversity of bacteriophages infecting Microbacterium spp
The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics
Models of classroom assessment for course-based research experiences
Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessmentâ(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learningâalong with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate studentsâ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education
Xuper the Host Hopper
Viruses have many different mechanisms that aid in the infection of their hosts. The blueprints of these mechanisms are encoded in the DNA of the virusâs genome, and often limit the virus to a specific host it can infect. Bacteriophages (phages), are viruses that infect bacteria. They are typically seen to only infect one specific host. Over the last several years, students at Illinois Wesleyan University have amassed a collection of about fifty different bacteriophages capable of infecting the bacterial host Rhodobacter capsulatus. While these phages are all unique, RcXuper has become one of the most interesting phages in our collection due to its ability to jump between bacterial hosts. The aim of this research is to identify the gene or genes that allow RcXuper to jump hosts, using a similar newly-isolated related phage, Aliso, that does not have the same capabilities. This research will contribute to the overall understanding of how viruses evolve and move into new hosts. It may also help identify ways to improve phage therapy, the use of phages to cure bacterial infections in order to prevent further antibiotic resistance (or as an alternative to antibiotics). Since phages tend to be highly specific, they have the potential to only rid a person of certain pathogens, but this high specificity can lead to problems if the bacteria gain resistance to the phage. Ultimately it is hoped that this research contributes to our understanding of the mechanisms through which phages can be genetically modified to control bacterial growth
Antiviral Properties of Fruit Juices on Bacteriophages
Viruses utilize many different mechanisms to infect their hosts and spread through populations. Because of this humans have long sought ways to treat viral infections and develop antiviral medications to stop these parasites or at least alleviate symptoms. Historically, a number of plant chemicals have been shown to have antimicrobial properties that have shown promise for these purposes. Prior to this study, cranberry juice has been demonstrated to be inhibitory for certain viruses. Over the last several years students at Illinois Wesleyan University have amassed a collection of about thirty different bacteriophages, viruses that infect bacteria. The aim of this research is to examine how widespread the effects of cranberry and other juices are on a variety of these bacteriophages. Bacteriophages from the Illinois Wesleyan University collection were exposed to different fruit juices to see how their replication was affected. The effects of juice on viral replication on a variety of virus types were examined and the results of these experiments will be discussed. This research has contributed to the overall knowledge of antivirals, and provides a new starting point for further research on the antiviral properties of juices
The Discovery and Characterization of Novel Bacteriophages Infecting Marine Bacterial Hosts
Bacteriophages are viruses that reproduce only when in the presence of a bacterial species susceptible to them. In the field of bacteriophage discovery, it is common practice to attempt isolations from fresh water or soil samples. However, there is a pronounced decline in the quantity of accessible data on sea water sample isolations. Currently, there is relatively little to no known information about bacteriophages that infect marine hosts related to the freshwater host Rhodobacter capsulatus. In recent years, researchers at Illinois Wesleyan University have amassed a collection of over 50 isolates capable of infecting the freshwater bacterium, R. capsulatus. The purpose of this research: to explore the largely under-studied universe of bacteriophages by discovering and isolating viruses that infect the marine bacterial strains Phaeobacter inhibens, Ruegeria pomeroyi, and Dinoroseobacter shibae. Three such bacteriophages were discovered; two that infect P. inhibens and one that infects D. shibae. Further testing determined if these bacteriophages are able to infect more than one species of bacteria previously listed, and a comparative analysis of genome sequences was conducted to determine the relation between previously found fresh water bacteriophages and the newly discovered marine bacteriophages. The results of these experiments combined with genomic analysis provides an insight into the world of marine bacteriophages and the evolution of viruses more broadly. In addition, this research may also contribute to the conceptualization of marine ecosystems as well as enhance the growing collection of bacteriophages that have potential use in bacterial infection treatment methods
Effects of Modified Phycobilin Biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002âż
The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity
Aliso and Jasper: Analysis of an Extraordinary Marine Phage and a Fun-Size Freshwater Phage
Bacteriophages, viruses that infect bacterial cells, are thought to be the most abundant and diverse biological entities on the planet. Due to an estimated 1031 bacteriophages in the world, they can be found in nearly every environment occupied by bacteria. Their great diversity allows for new phages to be discovered each time these environments are sampled. Through the isolation and propagation of individual specimens, our research group was able to characterize two distinct phages using polymerase chain reactions, transmission electron microscopy, and genomic sequencing. Bacteriophage Aliso was found to be notably unique due to its âcorn dogâ shaped capsid, long tail, and marine bacterial host Ruegeria pomeroyi. This phage also forms distinctly small plaques when cultured on its bacterial host, which is likely a result of having a relatively large genome size of 149,362bp. Our other phage, Jasper, infects the host Rhodobacter capsulatus, a freshwater bacterium. It is unique amongst phages isolated this past semester due to its small capsid size and short tail length. Unlike Aliso, Jasper has a relatively small genome of 41,414bp. Through analyzing phage genomes, new strains and adaptations can be uncovered, allowing for new phages to be clustered. Aliso however is an exception to this norm and is considered to be a singleton, as it does not fit into a cluster. Interestingly, it is most closely related to another phage named Xuper which was isolated on a freshwater host. Unlike Aliso, Jasper does cluster with other known phages. It most closely demonstrates similarities with one of the more divergent members of the RcC group, Kemmy. The similarities these phages share with each other and previously isolated phages will be presented
Lesions in Phycoerythrin Chromophore Biosynthesis in Fremyella diplosiphon Reveal Coordinated Light Regulation of Apoprotein and Pigment Biosynthetic Enzyme Gene Expression
We have characterized the regulation of the expression of the pebAB operon, which encodes the enzymes required for phycoerythrobilin synthesis in the filamentous cyanobacterium Fremyella diplosiphon. The expression of the pebAB operon was found to be regulated during complementary chromatic adaptation, the system that controls the light responsiveness of genes that encode several light-harvesting proteins in F. diplosiphon. Our analyses of pebA mutants demonstrated that although the levels of phycoerythrin and its associated linker proteins decreased in the absence of phycoerythrobilin, there was no significant modulation of the expression of pebAB and the genes that encode phycoerythrin. Instead, regulation of the expression of these genes is coordinated at the level of RNA accumulation by the recently discovered activator CpeR
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