Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).     

Extraction and long‐term storage of S‐layer proteins and flagella from Lysinibacillus sphaericus NCTC 9602: Building blocks for nanotechnology

Self‐assembling surface layer (SL) proteins of bacteria have been widely studied, in particular their use as molecularly defined, 2D coatings of technical surfaces. An important prerequisite is the availability of a sufficient amount of protein. However, a detailed and optimized protocol for the complete SL extraction is so far not available. Here, we describe the complete purification and reassembly procedure of an SL protein of Lysinibacillus sphaericus NCTC 9602, starting from the cultivation of cells, the preparation and purification of SL proteins up to the long‐term storage and in vitro self‐assembly of the proteins. All crucial steps of the procedure are assessed by different microscopic techniques, such as light microscopy, atomic force microscopy, and scanning electron microscopy as well as by SDS‐PAGE as a biochemical method. We demonstrate that storage of the protein in the presence of sodium azide or upon lyophilization allows the preservation of the self‐assembly properties for at least 9 years. Additionally, we describe a method allowing the extraction of intact flagella with lengths in the range up to 4 μm. Flagella may have applications in bio‐nanotechnology, for example as templates for metallic nanowires.     

Novel Mechanism of Arenavirus-Induced Liver Pathology

Viral hemorrhagic fevers (VHFs) encompass a group of diseases with cardinal symptoms of fever, hemorrhage, and shock. The liver is a critical mediator of VHF disease pathogenesis and high levels of ALT/AST transaminases in plasma correlate with poor prognosis. In fact, Lassa Fever (LF), the most prevalent VHF in Africa, was initially clinically described as hepatitis. Previous studies in non-human primate (NHP) models also correlated LF pathogenesis with a robust proliferative response in the liver. The purpose of the current study was to gain insight into the mechanism of liver injury and to determine the potential role of proliferation in LF pathogenesis. C57Bl/6J mice were infected with either the pathogenic (for NHPs) strain of lymphocytic choriomeningitis virus (LCMV, the prototypic arenavirus), LCMV-WE, or with the non-pathogenic strain, LCMV-ARM. As expected, LCMV-WE, but not ARM, caused a hepatitis-like infection. LCMV-WE also induced a robust increase in the number of actively cycling hepatocytes. Despite this increase in proliferation, there was no significant difference in liver size between LCMV-WE and LCMV-ARM, suggesting that cell cycle was incomplete. Indeed, cells appeared arrested in the G₁ phase and LCMV-WE infection increased the number of hepatocytes that were simultaneously stained for proliferation and apoptosis. LCMV-WE infection also induced expression of a non-conventional virus receptor, AXL-1, from the TAM (TYRO3/AXL/MERTK) family of receptor tyrosine kinases and this expression correlated with proliferation. Taken together, these results shed new light on the mechanism of liver involvement in VHF pathogenesis. Specifically, it is hypothesized that the induction of hepatocyte proliferation contributes to expansion of the infection to parenchymal cells. Elevated levels of plasma transaminases are likely explained, at least in part, by abortive cell cycle arrest induced by the infection. These results may lead to the development of new therapies to prevent VHF progression.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.