Bacteriophages and their structural organisation

Viruses are extremely small infectious particles that are not visible in a light microscope, and are able to pass through fine porcelain filters. They exist in a huge variety of forms and infect practically all living systems: animals, plants, insects and bacteria. All viruses have a genome, typically only one type of nucleic acid, but it could be one or several molecules of DNA or RNA, which is surrounded by a protective stable coat (capsid) and sometimes by additional layers which may be very complex and contain carbohydrates, lipids, and additional proteins. The viruses that have only a protein coat are named “naked”, or non- enveloped viruses. Many viruses have an envelope (enveloped viruses) that wraps around the protein capsid. This envelope is formed from a lipid membrane of the host cell during the release of a virus out of the cell

Applications of AFM in pharmaceutical sciences

Atomic force microscopy (AFM) is a high-resolution imaging technique that uses a small probe (tip and cantilever) to provide topographical information on surfaces in air or in liquid media. By pushing the tip into the surface or by pulling it away, nanomechanical data such as compliance (stiffness, Young’s Modulus) or adhesion, respectively, may be obtained and can also be presented visually in the form of maps displayed alongside topography images. This chapter outlines the principles of operation of AFM, describing some of the important imaging modes and then focuses on the use of the technique for pharmaceutical research. Areas include tablet coating and dissolution, crystal growth and polymorphism, particles and fibres, nanomedicine, nanotoxicology, drug-protein and protein-protein interactions, live cells, bacterial biofilms and viruses. Specific examples include mapping of ligand-receptor binding on cell surfaces, studies of protein-protein interactions to provide kinetic information and the potential of AFM to be used as an early diagnostic tool for cancer and other diseases. Many of these reported investigations are from 2011-2014, both from the literature and a few selected studies from the authors’ laboratories

A nano-view of West Nile virus-induced cellular changes during infection

BACKGROUND: Microscopic imaging of viruses and their interactions with and effects on host cells are frequently held back by limitations of the microscope's resolution or the invasive nature of the sample preparation procedures. It is also difficult to have a technique that would allow simultaneous imaging of both surface and sub-surface on the same cell. This has hampered endeavours to elucidate virus-host interactions. Atomic Force Microscopy (AFM), which is commonly used in the physical sciences, is now becoming a good correlative form of microscopy used to complement existing optical, confocal and electron microscopy for biological applications RESULTS: In this study, the West Nile (Sarafend) virus-infected Vero cell model was used. The atomic force microscope was found to be useful in producing high resolution images of virus-host events with minimal sample processing requirements. The AFM was able to image the budding of the West Nile (Sarafend) virus at the infected cell surface. Proliferation of the filopodia and thickening of clusters of actin filaments accompanied West Nile virus replication. CONCLUSIONS: The study shows that the AFM is useful for virus-host interaction studies. The technique provides morphological information on both the virus and the host cell during the infection stages

Physical effects of the respiratory syncytial virus on human cells.

Respiratory syncytial virus is the leading cause of lower respiratory tract infection in infants and currently lacks an effective vaccine or treatment beyond symptom relief. The atomic force microscope is particularly well suited for imaging biological samples including DNA, proteins, cells, and viral activity. Analyzing fixed HEp-2 cells with the AFM after infection and incubation with RSV for periods up to 24 hrs reveals several physical changes that may lead to a better understanding of the viral effects on living cells. Comparing cells fixed with methanol or glutaraldehyde and osmium tetroxide suggests substantial volume losses in the cells regardless of the fixatives used. Suggested mechanisms for the volume decrease include a combination of the creation of an osmotic pressure gradient within the cell and overall cellular collapse. Elastic modulus calculations were performed on the fixed cells at each duration of infection and analyzed for correlations to other physical properties. Hydrophobic interaction forces were also measured and analyzed in order to characterize chemical changes to the cell membrane. It was found that the elastic modulus did not decrease throughout the entire infection for up to 24 hours, but the hydrophobic expression on the cell surface steadily decreased over this same period. This decrease is attributed to the rapid removal and repair of the cell membrane by the RSV virions during exocytosis. Based on this study on methanol fixed cells, it was confirmed that RSV infection does not appear to modify the actin cytoskeleton of human cells

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized

The Boston University Photonics Center annual report 2013-2014

This repository item contains an annual report that summarizes activities of the Boston University Photonics Center in the 2013-2014 academic year. The report provides quantitative and descriptive information regarding photonics programs in education, interdisciplinary research, business innovation, and technology development. The Boston University Photonics Center (BUPC) is an interdisciplinary hub for education, research, scholarship, innovation, and technology development associated with practical uses of light.This annual report summarizes activities of the Boston University Photonics Center in the 2013–2014 academic year.This has been a good year for the Photonics Center. In the following pages, you will see that the center’s faculty received prodigious honors and awards, generated more than 100 notable scholarly publications in the leading journals in our field, and attracted $14.5M in new research grants and contracts this year. Faculty and staff also expanded their efforts in education and training, through National Science Foundation–sponsored sites for Research Experiences for Undergraduates and for Teachers. As a community, we hosted a compelling series of distinguished invited speakers, and emphasized the theme of Innovations at the Intersections of Micro/Nanofabrication Technology, Biology, and Biomedicine at our annual Future of Light Symposium. We took a leadership role in running national workshops on emerging photonic fields, including an OSA Incubator on Controlled Light Propagation through Complex Media, and an NSF Workshop on Noninvasive Imaging of Brain Function. Highlights of our research achievements for the year include a distinctive Presidential Early Career Award for Scientists and Engineers (PECASE) for Assistant Professor Xue Han, an ambitious new DoD-sponsored grant for Multi-Scale Multi-Disciplinary Modeling of Electronic Materials led by Professor Enrico Bellotti, launch of our NIH-sponsored Center for Innovation in Point of Care Technologies for the Future of Cancer Care led by Professor Cathy Klapperich, and successful completion of the ambitious IARPA-funded contract for Next Generation Solid Immersion Microscopy for Fault Isolation in Back-Side Circuit Analysis led by Professor Bennett Goldberg. These three programs, which represent more than$20M in research funding for the University, are indicative of the breadth of Photonics Center research interests: from fundamental modeling of optoelectronic materials to practical development of cancer diagnostics, from exciting new discoveries in optogenetics for understanding brain function to the achievement of world-record resolution in semiconductor circuit microscopy. Our community welcomed an auspicious cohort of new faculty members, including a newly hired assistant professor and a newly hired professor (and Chair of the Mechanical Engineering Department). The Industry/University Cooperative Research Center—the centerpiece of our translational biophotonics program—continues to focus on advancing the health care and medical device industries, and has entered its fourth year of operation with a strong record of achievement and with the support of an enthusiastic industrial membership base

Label-free microscopy for virus infections

Microscopy has been essential to elucidate micro- and nano-scale processes in space and time, and has provided insight into cell and organismic function. It is widely employed in cell biology, microbiology, physiology, clinical sciences and virology. While label-dependent microscopy, such as fluorescence microscopy, provides molecular specificity, it has remained difficult to multiplex in live samples. In contrast, label-free microscopy reports on overall features of the specimen at minimal perturbation. Here, we discuss modalities of label-free imaging at the molecular, cellular and tissue levels, including transmitted light microscopy, quantitative phase imaging, cryogenic electron microscopy or tomography, and atomic force microscopy. We highlight how label-free microscopy is used to probe the structural organization and mechanical properties of viruses, including virus particles and infected cells across a wide range of spatial scales. We discuss the working principles of imaging procedures and analyses, and showcase how they open new avenues in virology. Finally, we discuss orthogonal approaches that enhance and complement label-free microscopy techniques. Mini Abstract: Label-free imaging gives unprecedented insight into viruses at macroscopic, molecular and atomic levels. We present the major label-free imaging techniques and discuss how they are used for virus particles and infected cells. The power of label-free microscopy promises to enhance discovery of unknown aspects of infectious and therapeutic agents

Structural Organization of DNA in Chlorella Viruses

Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes