Expanding the Repertoire of Gene Tools for Precise Manipulation of the Clostridium difficile Genome: Allelic Exchange Using pyrE Alleles

Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the ‘correction’ method (5–7 days) makes pyrE ⁻ strains attractive hosts for mutagenesis studies.     

Dipole source localization by the mottled sculpin II. The role of lateral line excitation patterns

Extracellular, single unit recording techniques were used to measure the responses of posterior lateral line nerve fibers to a 50-Hz dipole source that slowly changed its location along the length of the fish. The flow-field equations for a dipole source were used to model the pressure gradient pattern and thus, the expected excitation pattern along a linear array of lateral line receptor organs for different source locations. Finally, excitation patterns were similarly modeled along the left and right side of the fish's head for actual steps taken by sculpin in approach pathways to the 50-Hz dipole source. Spatial histograms of posterior lateral line nerve fiber responses to different locations of the dipole source could be predicted from pressure gradient patterns modeled from the flow-field equations, confirming that the modeling approach applied to behavioral results was a good predictor of excitation patterns likely to be encoded by the lateral line periphery. An examination of how modeled excitation patterns changed from one position to the next in typical approach pathways and how patterns differed between positions from which successful and unsuccessful strikes were launched suggests that approach and strike strategies can indeed be explained by the information available in excitation patterns. In particular, changes in the spatial distribution of pressure gradient directions (polarities), available only when the source is lateral (as opposed to directly in front of the fish), appear to enhance the ability of sculpin to determine source distance. Without such information, misses are more likely to occur and successful strikes are more likely to be launched from short distances only. Accepted: 23 October 1996     

Journey to the centre of the cell: Virtual reality immersion into scientific data

Visualization of scientific data is crucial not only for scientific discovery but also to communicate science and medicine to both experts and a general audience. Until recently, we have been limited to visualizing the three‐dimensional (3D) world of biology in 2 dimensions. Renderings of 3D cells are still traditionally displayed using two‐dimensional (2D) media, such as on a computer screen or paper. However, the advent of consumer grade virtual reality (VR) headsets such as Oculus Rift and HTC Vive means it is now possible to visualize and interact with scientific data in a 3D virtual world. In addition, new microscopic methods provide an unprecedented opportunity to obtain new 3D data sets. In this perspective article, we highlight how we have used cutting edge imaging techniques to build a 3D virtual model of a cell from serial block‐face scanning electron microscope (SBEM) imaging data. This model allows scientists, students and members of the public to explore and interact with a “real” cell. Early testing of this immersive environment indicates a significant improvement in students’ understanding of cellular processes and points to a new future of learning and public engagement. In addition, we speculate that VR can become a new tool for researchers studying cellular architecture and processes by populating VR models with molecular data.      

Human Immunodeficiency Virus Neurotropism: an Analysis of Viral Replication and Cytopathicity for Divergent Strains in Monocytes and Microglia

Productive replication of human immunodeficiency virus type 1 (HIV-1) in brain macrophages and microglia is a critical component of viral neuropathogenesis. However, how virus-macrophage interactions lead to neurological disease remains incompletely understood. Possibly, a differential ability of virus to replicate in brain tissue macrophages versus macrophages in other tissues underlies HIV-1 neurovirulence. To these ends, we established systems for the isolation and propagation of pure populations of human microglia and then analyzed the viral life cycles of divergent HIV-1 strains in these cells and in cultured monocytes by using identical viral inocula and indicator systems. The HIV-1 isolates included those isolated from blood, lung tissue, cerebrospinal fluids (CSF), and brain tissues of infected subjects: HIV-1_ADA and HIV-1_89.6 (from peripheral blood mononuclear cells), HIV-1_DJV and HIV-1_JR-FL (from brain tissue), HIV-1_SF162 (from CSF), and HIV-1_BAL (from lung tissue). The synthesis of viral nucleic acids and viral mRNA, cytopathicity, and release of progeny virions were assessed. A significant heterogeneity among macrophage-tropic isolates for infection of monocytes and microglia was demonstrated. Importantly, a complete analysis of the viral life cycle revealed no preferential differences in the abilities of the HIV-1 strains tested to replicate in microglia and/or monocytes. Macrophage tropism likely dictates the abilities of HIV-1 to invade, replicate, and incite disease within its microglial target cells.     

Dietary Supplementation with S-Adenosyl Methionine was Associated with Protracted Reduction of Seizures in a Line of Transgenic Mice

Transgenic mice, although useful for analyses of gene function, can present unanticipated phenotypic manifestations, including behavioral problems, that may not be directly associated with the gene of interest but rather due to the complex interplay inherent in genomes. These unexpected events can present unique insight into gene function, leading to an advantage in some situations, yet in others can confound interpretation and compromise usefulness of the transgenic line. Here we document that short-term supplementation with S-adenosyl methionine (SAM)—a nutriceutical known to regulate neurotransmitter levels, improve working memory, and reduce aggression—reduced handling- and startling-induced seizures that otherwise precluded behavioral analyses in a transgenic line. This effect lasted for at least 1 mo after withdrawal of SAM and allowed mice to be used in standard maze analyses. These findings suggest that short-term administration of a neurotropic nutriceutical may provide a functional rescue for behavioral studies in an otherwise intractable transgenic mouse line as well as improve the welfare of similar lines.Abbreviations: SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionineSite-directed mutagenesis, gene deletion, and the insertion of exogenous genes present powerful tools for genetic analyses. Incorporation of such genetic alterations into the murine germline, and the resultant generation of transgenic strains of mice, has provided novel insight into the roles of genes, and their interaction with other genes, at all stages of life. However, transgenic mice can present unanticipated behavioral problems that may not be associated directly with the gene of interest but rather are due to the complex interplay inherent in genomes.²⁰ This unexpected outcome can present unique insight into gene function, leading to an advantage in some situations, yet in others can confound interpretation and compromise usefulness of the transgenic line. Genetic variability of inbred strains can confound interpretation of behavior,¹⁸ especially if behavioral analyses are part of the regimen to be studied.²⁶The presence of 1 or more ApoE4 alleles is associated with an increased risk of Alzheimer disease.¹³ Transgenic mice in which the single murine ApoE allele has been ablated and replaced with human apolipoprotein E isoforms have been useful models for studying the impact of ApoE on age-related cognitive decline and Alzheimer disease. In addition to impaired cognition, mice lacking murine ApoE and expressing human ApoE4 (ApoE4 mice) display increased aggression as compared with normal mice, mice lacking murine ApoE, or mice lacking murine ApoE and expressing other human ApoE alleles.^5,6 These behavioral manifestations of ApoE4 mice are useful in that Alzheimer disease is often accompanied by behavioral trauma, including pyschosis and agitation,⁷ and ApoE4 has been associated with an increase psychotic symptoms in humans.²⁷Recent shipments of ApoE4 mice displayed violent spontaneous and handling-induced convulsions (hereafter referred to as seizures for the sake of simplicity), which included jumping and eventual prostration due to overt exhaustion. These seizures occurred regardless of how quietly or slowly the handler or caregiver moved. These seizures, which persisted for more than 1 mo, had not previously been observed in ApoE4 mice or other mice in our facility and precluded the intended use of the ApoE4 mice in standard maze trials.⁵In our ongoing studies, we had observed that dietary supplementation with the nutriceutical S-adenosyl methionine (SAM) reduced aggressive behavior in ApoE4 mice.⁵ SAM also restores neurotransmitter balance, increases working memory, and modulates neuronal activity.^6,8,17,19 We therefore hypothesized that SAM supplementation would reduce or alleviate handling-induced seizures. Here we document that short-term administration of SAM reduced of seizures for extended periods, to the extent that these mice could be used in behavioral studies. We discuss the possibility that such an approach may be useful for habituation of other mouse lines displaying similar behavior difficulties.     

Generic plasmid DNA production platform incorporating low metabolic burden seed‐stock and fed‐batch fermentation processes

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is “plugged” into a validated vector, and rapidly produced in a validated, fermentation—purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably “toxic” or otherwise low yielding in standard fermentation processes. We report cell bank and fermentation process unit operation innovations that reduce plasmid‐mediated metabolic burden, enabling successful production of previously known toxic influenza hemagglutinin antigen genes. These processes, combined with vector backbone modifications, doubled fermentation productivity compared to existing high copy vectors, such as pVAX1 and gWiz, resulting in high plasmid yields (up to 2,220 mg/L, 5% of total dry cell weight) even with previously identified toxic or poor producing inserts. Biotechnol. Bioeng. 2009;103: 1129–1143. © 2009 Wiley Periodicals, Inc.