An investigation of <Emphasis Type="Italic">Clostridium</Emphasis> species present in nutraceutical preparations of <Emphasis Type="Italic">Arthrospira platensis</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) for human consumption

The presence of the anaerobic spore former Clostridium in Arthrospira platensis destined for human consumption is generally not assessed during quality assurance procedures. As this nutraceutical is administered as complementary medicine to the immunocompromised, this study aimed to investigate the presence of these potential pathogens. Anaerobic counts performed on tablets from a single manufacturer indicated an excess of 10⁵ CFU/endospores g⁻¹ tablet for three different A. platensis batches. Tests for coliforms for use as “indicators” of pathogens in the tablets were negative. Using classic culture techniques, five species of Clostridium were isolated. Subsequent use of PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting of tablets showed a divergent microbial population, with a predominance of anaerobic endospore formers, including Clostridium. Sequencing of a 1.5 kb 16S rDNA clone library and phylogenetic analyses of prominent operational taxonomic units confirmed the presence of an additional five Clostridium spp. and other genera in the tablets. A composite molecular ladder, using 16S rRNA DGGE amplicons of 17 representative bacterial species was constructed to assist in identifying anaerobes present in tablets sourced from three different A. platensis manufacturers. Results indicated that commercial A. platensis preparations were contaminated with potentially hazardous clostridia and other anaerobic species. Results suggest that certain commercial A. platensis preparations require stringent microbial quality assurance measures to ensure safe use as a nutraceutical for the immunocompromised and the general public.     

Control of mRNA export by adenovirus E4orf6 and E1B55K proteins during productive infection requires E4orf6 ubiquitin ligase activity

During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6⁻ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.     

Oxylipin analysis methods

Practical guidelines for monitoring and measuring compounds such as jasmonates, ketols, ketodi(tri)enes and hydroxy-fatty acids as well as detecting the presence of novel oxylipins are presented. Additionally, a protocol for the penetrant analysis of non-enzymatic lipid oxidation is described. Each of the methods, which employ gas chromatography/mass spectrometry, can be applied without specialist knowledge or recourse to the latest analytical instrumentation. Additional information on oxylipin quantification and novel protocols for preparing oxygen isotope-labelled internal standards are provided. Four developing areas of research are identified: (i) profiling of the unbound cellular pools of oxylipins; (ii) profiling of esterified oxylipins and/or monitoring of their release from parent lipids; (iii) monitoring of non-enzymatic lipid oxidation; (iv) analysis of unstable and reactive oxylipins. The methods and protocols presented herein are designed to give technical insights into the first three areas and to provide a platform from which to enter the fourth area.     

The blockade of endothelin A receptor protects astrocytes against hypoxic injury: common effects of BQ-123 anderythropoietin on the rejuvenation of the astrocyte population

In the present study the role of endothelin (ET) and its receptors (ETA-R and ETB-R) in cellular mechanisms underlying the resistance of astroglial cells to low oxygen level and development of hypoxia has been investigated. To define the influences of ET and its receptors on survival and on antigenic as well as morphologic differentiation of rat astroglial cells in normoxic (NC) and hypoxic culture (HC) the selective antagonists of ETA-R (BQ-123) and ETB-R (BQ-788) were used. Treatment of HC with BQ-123 caused an increase in cell number and inhibited the hypoxia-induced apoptosis by 37%. BQ-123 decreased the hypoxia-induced cytotoxicity in HC. These effects of BQ-123 were abolished in cultures simultaneously treated with BQ-123 and BQ-788. Administration of BQ-788 alone decreased the number of living cells in NC, but not in HC. The activity of caspase-3/-7 was not changed by exposure of NC and HC to BQ-788. The protection provided by BQ-123 to astroglial cells against cytotoxicity in NC and HC was similar to that of erythropoietin (EPO), a cytokine with established neuroprotective effects. The functional improvement of astroglial cells and slowing down of their differentiation under exposure to BQ-123, or EPO, or BQ-123 + EPO has been evidenced by an increased number of nestin+/glial fibrillary acidic protein-positive (GFAP+) astrocytes accompanied by decrease of nestin-/GFAP+ cells. The simultaneous treatment with BQ-123 and EPO additionally decreased the activities of caspase-3/-7 (64%) and release of LDH into the medium (94%). The benefits in the functional states of astrocytes obtained by combined treatment of HC with BQ-123 and EPO suggest a new therapeutic strategy in treatment of hypoxic brain injury.     

Effect of pathogenic mutations on the structure and dynamics of Alzheimer’s Aβ42-amyloid oligomers

Converging lines of evidence suggest that soluble Aβ-amyloid oligomers play a pivotal role in the pathogenesis of Alzheimer’s disease, and present direct effectors of synaptic and cognitive dysfunction. Three pathological E22-Aβ-amyloid point mutants (E22G, E22K, E22Q) and the deletion mutant E22Δ exhibit an enhanced tendency to form prefibrillar aggregates. The present study assessed the effect of these four mutations using molecular dynamics simulations and subsequent structural and energetic analyses. Our data shows that E22 plays a unique role in wild type Aβ, since it has a destabilising effect on the oligomer structure due to electrostatic repulsion between adjacent E22 side chains. Mutations in which E22 is replaced by an uncharged residue result in higher oligomer stability. This effect is also observed to a lesser extent for the E22K mutation and is consistent with its lower pathogenicity compared to other mutants. Interestingly, deletion of E22 does not destroy the amyloid fold but is compensated by local changes in the backbone geometry that allow the preservation of a structurally important salt bridge. The finding that all mutant oligomers investigated exhibit higher internal stability than the wild type offers an explanation for the experimentally observed enhanced oligomer formation and stability.     

Is the degree of clonality of forest herbs dependent on gap age? Using fingerprinting approaches to assess optimum successional stages for montane forest herbs

Using molecular fingerprinting (amplified fragment length polymorphism [AFLP] method), we explored the potential of small-scale population analysis for understanding colonization patterns of herb layer species in forests after canopy disturbance. We investigated three common forest understorey species with different life forms (Trientalis europaea, Calamagrostis villosa, and Vaccinium myrtillus) in the Harz Mountains in Germany in three different gap age classes and undisturbed forest. For two of them (T. europaea and C. villosa), we analyzed clone sizes and clonal structure. We hypothesized that clone sizes depend on age since gap formation and are affected by light availability. Mean patch sizes of V. myrtillus, T. europaea, and C. villosa formed were 3.7 m(2), 27.9 m(2), and 40.6 m(2), respectively. Trientalis europaea and C. villosa patches consisted mostly of more than one genet. Largest clone sizes of T. europaea were encountered in gaps of intermediate successional age (15-60 years, averaged minimum estimation of clone sizes: 6.56 m(2)) whereas clone size of C. villosa was found to be independent from gap age and had a mean minimum clone size of 0.49 m(2). In both species, clone size was positively related to light availability. Additionally, there was a positive relationship between clone size and ramet density for T. europaea and C. villosa. Genetic variation was higher within populations of T. europaea and C. villosa than among populations. Trientalis europaea was the only species with a clear genetic isolation by distance, pointing at an equilibrium between gene flow and genetic drift. In conclusion, we showed that forest canopy gap dynamics clearly affect the small-scale structure of populations of understorey plants. Species with high lateral growth rates, such as T. europaea offer the possibility to serve as "ecological clock" for dating ecological processes.     

Antigen-dependent and -independent mechanisms of T and B cell hyperactivation during chronic HIV-1 infection

Continuous loss of CD4(+) T lymphocytes and systemic immune activation are hallmarks of untreated chronic HIV-1 infection. Chronic immune activation during HIV-1 infection is characterized by increased expression of activation markers on T cells, elevated levels of proinflammatory cytokines, and B cell hyperactivation together with hypergammaglobulinemia. Importantly, hyperactivation of T cells is one of the best predictive markers for progression toward AIDS, and it is closely linked to CD4(+) T cell depletion and sustained viral replication. Aberrant activation of T cells is observed mainly for memory CD4(+) and CD8(+) T cells and is documented, in addition to increased expression of surface activation markers, by increased cell cycling and apoptosis. Notably, the majority of these activated T cells are neither HIV specific nor HIV infected, and the antigen specificities of hyperactivated T cells are largely unknown, as are the exact mechanisms driving their activation. B cells are also severely affected by HIV-1 infection, which is manifested by major changes in B cell subpopulations, B cell hyperactivation, and hypergammaglobulinemia. Similar to those of T cells, the mechanisms underlying this aberrant B cell activation remain largely unknown. In this review, we summarized current knowledge about proposed antigen-dependent and -independent mechanisms leading to lymphocyte hyperactivation in the context of HIV-1 infection.     

Biofilm formation by Salmonella enterica serovar Typhimurium colonizing solid tumours

Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella-colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.     

Antibody production in Bacillus megaterium: strategies and physiological implications of scaling from microtiter plates to industrial bioreactors

Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5-100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH(4))(2)SO(4), and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.