Intracellular assembly of cyanophage Syn5 proceeds through a scaffold-containing procapsid

Syn5 is a marine cyanophage that is propagated on the marine photosynthetic cyanobacterial strain Synechococcus sp. WH8109 under laboratory conditions. Cryoelectron images of this double-stranded DNA (dsDNA) phage reveal an icosahedral capsid with short tail appendages and a single novel hornlike structure at the vertex opposite the tail. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. The one-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ～45 min, a latent phase of ～60 min, and a burst size of 20 to 30 particles per cell at 28°C. SDS-PAGE and Western blot analysis of cell lysates at different times after infection showed the synthesis of major virion proteins and their increase as the infection progressed. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. It migrated anomalously on SDS-PAGE, similar to the phage T7 scaffolding protein. Particles lacking DNA but containing the coat and scaffolding proteins were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA but filled with protein density, presumably scaffolding protein. These findings suggest that the cyanophages form infectious virions through the initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid-mediated assembly pathway may have originated with the cyanophages.     

The glucose transporter GLUT1 and the tight junction protein occludin in nasal olfactory mucosa.

The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.     

Both TRIF and IPS-1 Adaptor Proteins Contribute to the Cerebral Innate Immune Response against Herpes Simplex Virus 1 Infection

Toll-like receptors (TLRs) and RNA helicases (RLHs) are important cell sensors involved in the immunological control of viral infections through production of type I interferon (IFN). The impact of a deficiency in the TRIF and IPS-1 adaptor proteins, respectively, implicated in TLR3 and RLH signaling pathways, was investigated during herpes simplex virus 1 (HSV-1) encephalitis. TRIF^−/−, IPS-1^−/−, and C57BL/6 wild-type (WT) mice were infected intranasally with 7.5 × 10⁵ PFU of HSV-1. Mice were monitored for neurological signs and survival over 20 days. Groups of mice were sacrificed on days 3, 5, 7, 9, and 11 postinfection for determination of brain viral replication by quantitative PCR (qPCR), plaque assay, and immunohistochemistry and for alpha/beta interferon (IFN-α/β) levels and phosphorylation of interferon regulatory factors 3 and 7 (IRF-3 and -7) in brain homogenates by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. TRIF^−/− and IPS-1^−/− mice had higher mortality rates than WT mice (P = 0.02 and P = 0.09, respectively). Viral antigens were more disseminated throughout the brain, correlating with a significant increase in brain viral load for TRIF^−/− (days 5 to 9) and IPS-1^−/− (days 7 and 9) mice compared to results for the WT. IFN-β production was reduced in brain homogenates of TRIF^−/− and IPS-1^−/− mice on day 5 compared to results for the WT, whereas IFN-α levels were increased on day 7 in TRIF^−/− mice. Phosphorylation levels of IRF-3 and IRF-7 were decreased in TRIF^−/− and IPS-1^−/− mice, respectively. These data suggest that both the TRIF and IPS-1 signaling pathways are important for the control of HSV replication in the brain and survival through IFN-β production.     

Novel Fatty Acid Modifications of Transportan 10

Cell-penetrating peptides (CPPs) are able to efficiently internalize into cells and can therefore be used as vectors for non-viral cellular delivery of different cargoes. Previous studies have shown that hydrophobic modifications of different CPPs can increase their transfection efficiency dramatically. In this study we have modified the cell penetrating-peptide transportan 10 (TP10) with a variety of hydrophobic molecules to determine the role of hydrophobicity in the uptake of these molecules. The results can be used to synthesize more efficient delivery vectors. To evaluate how these constructs are able to transport cargoes into cells we used 2′-OMe splice correcting oligonucleotides. Non-covalent peptide-cargo complexes were formed and their transfection efficiency was measured using a luciferase readout system. The hydrophobicity of the novel modifications was correlated with their biological efficacy. We determined the most efficient range of hydrophobicity for TP10 analogs for delivering oligonucleotides into cells. In order to assess how the transfection efficacy of these particles is dependent on their size the hydrodynamic diameter of the formed nanoparticles was measured using dynamic light scattering. These findings will be used to develop highly efficient non-viral gene therapy vectors.     

Mammalian cell retention devices for stirred perfusion bioreactors

Within the spectrum of current applications for cell culture technologies, efficient large-scale mammalian cell production processes are typically carried out in stirred fed-batch or perfusion bioreactors. The specific aspects of each individual process that can be considered when determining the method of choice are presented. A major challenge for perfusion reactor design and operation is the reliability of the cell retention device. Current retention systems include cross-flow membrane filters, spin-filters, inclined settlers, continuous centrifuges and ultrasonic separators. The relative merits and limitations of these technologies for cell retention and their suitability for large-scale perfusion are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil,shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.     

Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol

Regulatory T cells (Tregs) are a promising therapy for several immune-mediated conditions but manufacturing a homogeneous and consistent product, especially one that includes cryopreservation, has been challenging. Discarded pediatric thymuses are an excellent source of therapeutic Tregs with advantages including cell quantity, homogeneity and stability. Here we report systematic testing of activation reagents, cell culture media, restimulation timing and cryopreservation to develop a Good Manufacturing Practice (GMP)–compatible method to expand and cryopreserve Tregs. By comparing activation reagents, including soluble antibody tetramers, antibody-conjugated beads and artificial antigen-presenting cells (aAPCs) and different media, we found that the combination of Dynabeads Treg Xpander and ImmunoCult-XF medium preserved FOXP3 expression and suppressive function and resulted in expansion that was comparable with a single stimulation with aAPCs. Cryopreservation tests revealed a critical timing effect: only cells cryopreserved 1–3 days, but not >3 days, after restimulation maintained high viability and FOXP3 expression upon thawing. Restimulation timing was a less critical process parameter than the time between restimulation and cryopreservation. This systematic testing of key variables provides increased certainty regarding methods for in vitro expansion and cryopreservation of Tregs. The ability to cryopreserve expanded Tregs will have broad-ranging applications including enabling centralized manufacturing and long-term storage of cell products.     

Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136-147, 1997.