Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.     

Biochemical correlates to cortical dysplasia,gliosis, and astrocytoma infiltration in human epileptogenic cortex

The study provides detailed biochemical correlates to the common histopathological diagnoses in epilepsy. A dot immunobinding procedure was used for quantification of NSE, GFA, S-100, NCAM, NF 68 and NF 200. The material consisted of samples from 48 patients either selected for surgical treatment of partial epilepsy or for disorders not related to epilepsy. The histopthological diagnosis of the epileptic cases was: MCD (mild cortical dysplasia, microdysgenesis), gliosis, astrocytoma, ganglioglioma, oligodendroglioma and single cases. The concentration in non-epileptic white matter, in per cent of that in grey matter was: NSE, 85; GFA, 175; S-100, 117; NCAM, 43; NF 68,227 and NF 200, 173. The concentration of NSE as well as of GFA was close to normal in the specimens of the MCD and gliosis groups and of one subgroup of the astrocytomas. There was a striking inverse relationship of the GFA vs the NSE concentrations in the whole material. The concentrations of S-100 showed no such inverse relationship to NSE levels. In all the epileptic groups, total NCAM was lower than 50% of that of the non-epileptic group. The mean NF 68 and NF 200 concentration in the gliosis and astrocytoma groups was 75% of the of the non-epileptic group while the corresponding value for the MCD group was 50%. There was a positive correlation of immunochemically determined GFA and the histopathological gliosis score in the samples of epileptogenic cortex. There was no correlation between the concentration of GFA in the samples and the duration of epilepsy. The concentration of neuronal markers was relatively unaffected in the cortex of most patients with epilepsy related to MCD, gliosis and even to astrocytoma infiltration, even after years of seizures.Special issue dedicated to Dr. Claude Baxter.     

Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization

Spreading depolarization (SD) is a feed‐forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn²⁺ release and intracellular accumulation occurs with SD, and elevated intracellular Zn²⁺ ([Zn²⁺]_i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn²⁺]_i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn²⁺]_i loading prior to SD, by transient ZnCl₂ application with the Zn²⁺ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post‐synaptic potentials (fEPSPs) in a concentration‐dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn²⁺]_i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn²⁺ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn²⁺]_i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn²⁺]_i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn²⁺]_i accumulation in a range of brain injury models.     

Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.     

Integrating trait‐based empirical and modeling research to improve ecological restoration

A global ecological restoration agenda has led to ambitious programs in environmental policy to mitigate declines in biodiversity and ecosystem services. Current restoration programs can incompletely return desired ecosystem service levels, while resilience of restored ecosystems to future threats is unknown. It is therefore essential to advance understanding and better utilize knowledge from ecological literature in restoration approaches. We identified an incomplete linkage between global change ecology, ecosystem function research, and restoration ecology. This gap impedes a full understanding of the interactive effects of changing environmental factors on the long‐term provision of ecosystem functions and a quantification of trade‐offs and synergies among multiple services. Approaches that account for the effects of multiple changing factors on the composition of plant traits and their direct and indirect impact on the provision of ecosystem functions and services can close this gap. However, studies on this multilayered relationship are currently missing. We therefore propose an integrated restoration agenda complementing trait‐based empirical studies with simulation modeling. We introduce an ongoing case study to demonstrate how this framework could allow systematic assessment of the impacts of interacting environmental factors on long‐term service provisioning. Our proposed agenda will benefit restoration programs by suggesting plant species compositions with specific traits that maximize the supply of multiple ecosystem services in the long term. Once the suggested compositions have been implemented in actual restoration projects, these assemblages should be monitored to assess whether they are resilient as well as to improve model parameterization. Additionally, the integration of empirical and simulation modeling research can improve global outcomes by raising the awareness of which restoration goals can be achieved, due to the quantification of trade‐offs and synergies among ecosystem services under a wide range of environmental conditions.     

Factors influencing depth distribution of soft bottom inhabiting Laminaria saccharina (L.) Lamour. in Kiel Bay,Western Baltic

The kelp Laminaria saccharina dominates soft bottoms in 4–10 m depth in Kiel Bay. Experimental sporophytes transplanted to 2 and 5 m depth showed the typical annual growth pattern of Laminaria species. Surprisingly, 2 m plants died after the first resting phase, whereas 5 m plants survived and showed outgrowth of a new blade generation. Thalli at both depths were infected with the brown algal endophyte Streblonema aecidioides, with host deformations being significantly stronger in 2 m plants. Growth rates of infected sporophytes were reduced. Exclusion of UV light in 2 m depth resulted in less infected thalli. Discs excised from L. saccharina and cultivated in different photon fluence rates from 10–600 µmol m^–2 s^–1 did not differ in growth rate, photosynthesis or dark respiration. Hence, an exclusion of L. saccharina from shallow depths caused by high light cannot be concluded. We suggest the biological interaction with the endophyte S. aecidioides, amplified by UV light, to be most important for the exclusion of L. saccharina from shallow depths in the western Baltic.     

Streblonema (Ectocarpales,Phaeophyceae) infection in the kelp Laminaria saccharina (Laminariales,Phaeophyceae) in the western Baltic

The brown alga Laminaria saccharina is the dominant subtidal macroalga in Kiel Bay, western Baltic. It is infected by the microscopic brown alga, Streblonema aecidioides. Infected thalli may show symptoms of Streblonema disease, i.e. alterations of blade and stipe, ranging from dark spots to heavy deformations and completely crippled thalli. Samples taken from a single locality all year round show that (i) the host population is infected at a high rate of 87±13% (SD), but that (ii) a considerable proportion of thalli containing Streblonema does not show disease symptoms, and that (iii) juvenile hosts, which mainly appear in autumn, are infected at almost the same rate. Thus the infection seems to occur early in the host's life. Juveniles in nature show fewer symptoms of the disease than adults. Two months after infection, oxygen production and growth in laboratory-raised experimentally infected juvenile hosts was not different from uninfected controls. Experimental thalli showed more severe morphological alterations than uninfected controls only four months after infection. Both field and laboratory observations indicate that a lag phase exists between infection and outbreak of the disease.     

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.