The Gut of the Soil Microarthropod Folsomia candida (Collembola) Is a Frequently Changeable but Selective Habitat and a Vector for Microorganisms

Interaction potentials between soil microarthropods and microorganisms were investigated with Folsomia candida (Insecta, Collembola) in microcosm laboratory experiments. Microscopic analysis revealed that the volumes of the simple, rod-shaped guts of adult specimens varied with their feeding activity, from 0.7 to 11.2 nl. A dense layer of bacterial cells, associated with the peritrophic membrane, was detected in the midgut by scanning electron microscopy. Depending on the molting stage, which occurred at intervals of approximately 4 days, numbers of heterotrophic, aerobic gut bacteria changed from 4.9 × 10² to 2.3 × 10⁶ CFU per specimen. A total of 11 different taxonomic bacterial groups and the filamentous fungus Acremonium charticola were isolated from the guts of five F. candida specimens. The most abundant isolate was related to Erwinia amylovora (96.2% DNA sequence similarity to its 16S rRNA gene). F. candida preferred to feed on Pseudomonas putida and three indigenous gut isolates rather than eight different type culture strains. When luciferase reporter gene-tagged bacterial strains were pulse fed to F. candida, gut isolates were continuously shed for 8 days to several weeks but Escherichia coli HB101 was shed for only 1 day. Ratios of ingested to released bacterial cells demonstrated that populations of nonindigenous gut bacteria like Sinorhizobium meliloti L33 and E. coli HB101 were reduced by more than 4 orders of magnitude but that the population of gut isolate Alcaligenes faecalis HR4 was reduced only 500-fold. This work demonstrates that F. candida represents a frequently changeable but selective habitat for bacteria in terrestrial environments and that microarthropods have to be considered factors that modify soil microbial communities.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Hexon Modification to Improve the Activity of Oncolytic Adenovirus Vectors against Neoplastic and Stromal Cells in Pancreatic Cancer

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.     

Phylogenetic position of Gluconobacter species as a coherent cluster separated from all Acetobacter species on the basis of 16S ribosomal RNA sequences

Abstract The 16S rRNA sequences from the Gluconobacter species G. asaii G. cerinus and G. frateurii were determined and compared with homologous sequences from published databases and sequences of G. oxydans and Acetobacter species previously described [Sievers M., Ludwig W. and Teuber M. (1994) System. Appl. Microbiol. 17, 189–196]. The Gluconobacter species have unique 16S rRNA sequences and exhibit sequence similarity values of 97.4 to 99.1%, corresponding to 36 to 14 base differences. The phylogenetic tree inferring methods (distance matrix, maximum parsimony and maximum likelihood) show that the species of Gluconobacter form a coherent, closely related cluster. Based on the distance matrix method including Rhodopila globiformis as an outgroup reference organism, Gluconobacter is well separated from Acetobacter .     

H2O2 destruction by ascorbate-dependent systems from chloroplasts.

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3'-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated. System A is membrane-bound, reacts with 3,3'-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide. The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide. Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H2O2 which is produced in these organelles by photosynthetic O2 reduction.     

Multiple Stressors in Agricultural Streams: A Mesocosm Study of Interactions among Raised Water Temperature,Sediment Addition and Nutrient Enrichment

Changes to land use affect streams through nutrient enrichment, increased inputs of sediment and, where riparian vegetation has been removed, raised water temperature. We manipulated all three stressors in experimental streamside channels for 30 days and determined the individual and pair-wise combined effects on benthic invertebrate and algal communities and on leaf decay, a measure of ecosystem functioning. We added nutrients (phosphorus+nitrogen; high, intermediate, natural) and/or sediment (grain size 0.2 mm; high, intermediate, natural) to 18 channels supplied with water from a nearby stream. Temperature was increased by 1.4°C in half the channels, simulating the loss of upstream and adjacent riparian shade. Sediment affected 93% of all biological response variables (either as an individual effect or via an interaction with another stressor) generally in a negative manner, while nutrient enrichment affected 59% (mostly positive) and raised temperature 59% (mostly positive). More of the algal components of the community responded to stressors acting individually than did invertebrate components, whereas pair-wise stressor interactions were more common in the invertebrate community. Stressors interacted often and in a complex manner, with interactions between sediment and temperature most common. Thus, the negative impact of high sediment on taxon richness of both algae and invertebrates was stronger at raised temperature, further reducing biodiversity. In addition, the decay rate of leaf material (strength loss) accelerated with nutrient enrichment at ambient but not at raised temperature. A key implication of our findings for resource managers is that the removal of riparian shading from streams already subjected to high sediment inputs, or land-use changes that increase erosion or nutrient runoff in a landscape without riparian buffers, may have unexpected effects on stream health. We highlight the likely importance of intact or restored buffer strips, both in reducing sediment input and in maintaining cooler water temperatures.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.