An arginine switch in the species B adenovirus knob determines high-affinity engagement of cellular receptor CD46

Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.     

A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Growth Efficiency and Carbon Balance for the Sponge Haliclona oculata

To obtain more knowledge about carbon requirements for growth by sponges, the growth rate, respiration rate, and clearance rate was measured in situ in Haliclona oculata. We found that only 34% of the particulate carbon pumped through the sponge was used for both respiration and growth. The net growth efficiency, being the ratio of carbon incorporated in biomass and the total carbon used by the sponge for respiration and growth, was found to be 0.099 ± 0.013. Thus, about 10% of the total used carbon was fixed in biomass, and over 90% was used for generating energy for growth, maintenance, reproduction, and pumping. H. oculata had 2.5 μmol C available for every micromole O₂ consumed. A value of 0.75 for respiratory quotient (RQ in micromole CO₂ micromole O₂⁻¹) was used for H. oculata, which is the average value reported in literature for different marine invertebrates. Thus, carbon was available in excess to meet the respiratory demand. Oxygen was found not to be the limiting factor for growth, since only 3.3% of the oxygen pumped through the sponge body was used. Our results indicate that both oxygen and carbon availability are not limiting. The low growth efficiency agrees with the low growth rates found for the species used in this study.     

Biomarker discovery in heterogeneous tissue samples -taking the in-silico deconfounding approach

Background  

For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues.     

4-substituted cyclohexyl sulfones as potent, orally active gamma-secretase inhibitors

The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).     

Photosynthetic efficiency of Chlorella sorokiniana in a turbulently mixed short light‐path photobioreactor

To be able to study the effect of mixing as well as any other parameter on productivity of algal cultures, we designed a lab‐scale photobioreactor in which a short light path (SLP) of (12 mm) is combined with controlled mixing and aeration. Mixing is provided by rotating an inner tube in the cylindrical cultivation vessel creating Taylor vortex flow and as such mixing can be uncoupled from aeration. Gas exchange is monitored on‐line to gain insight in growth and productivity. The maximal productivity, hence photosynthetic efficiency, of Chlorella sorokiniana cultures at high light intensities (1,500 μmol m^?1 s^?1) was investigated in this Taylor vortex flow SLP photobioreactor. We performed duplicate batch experiments at three different mixing rates: 70, 110, and 140 rpm, all in the turbulent Taylor vortex flow regime. For the mixing rate of 140 rpm, we calculated a quantum requirement for oxygen evolution of 21.2 mol PAR photons per mol O₂ and a yield of biomass on light energy of 0.8 g biomass per mol PAR photons. The maximal photosynthetic efficiency was found at relatively low biomass densities (2.3 g L^?1) at which light was just attenuated before reaching the rear of the culture. When increasing the mixing rate twofold, we only found a small increase in productivity. On the basis of these results, we conclude that the maximal productivity and photosynthetic efficiency for C. sorokiniana can be found at that biomass concentration where no significant dark zone can develop and that the influence of mixing‐induced light/dark fluctuations is marginal. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Power consumption and maximum energy dissipation in a milliliter‐scale bioreactor

Mean power consumption and maximum local energy dissipation were measured as function of operating conditions of a milliliter‐scale stirred tank bioreactor (V = 12 mL) with a gas‐inducing impeller. A standard laboratory‐scale stirred tank bioreactor (V = 1,200 mL) with Rushton turbines was used as reference. The measured power characteristics (Newton number as function of Reynolds number) were the same on both scales. The changeover between laminar and turbulent flow regime was observed at a Reynolds number of 3,000 with the gas‐inducing stirrer on a milliliter‐scale. The Newton number (power number) in the turbulent flow regime was 3.3 on a milliliter‐scale, which is close to values reported for six‐blade Rushton turbines of standard bioreactors. Maximum local energy dissipation (ε_max) was measured using a clay/polymer flocculation system. The maximum local energy dissipation in the milliliter‐scale stirred tank bioreactor was reduced compared with the laboratory‐scale stirred tank at the same mean power input per unit mass (ε_ø), yielding ε_max/ε_ø ≈ 10 compared with ε_max/ε_ø ≈ 16. Hence, the milliliter‐scale stirred tank reactor distributes power more uniformly in the reaction medium. These results are in good agreement with literature data, where a decreasing ε_max/ε_ø with increasing ratio of impeller diameter to reactor diameter is found (d/D = 0.7 compared with d/D = 0.4). Based on these data, impeller speeds can now be easily adjusted to achieve the same maximum local energy dissipation at different scales. This enables a more reliable and robust scale‐up of bioprocesses from milliliter‐scale to liter‐scale reactors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010