EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.     

On the observation of a gem diol intermediate after O–O bond cleavage by extradiol dioxygenases. A hybrid DFT study

Catalytic cycle intermediates of a representative extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), have recently been characterized in crystallo by Kovaleva and Lipscomb. The structures of the identified species indicate that the process of inserting oxygen into the catechol ring occurs stepwise, and involves an Fe(II)-alkylperoxo intermediate and its O–O cleavage product: a gem diol species. In general, these findings corroborate the results of our previous computational studies; however, the fact that the gem diol species is stable enough to be observed in the crystal form seems to be at odds with the computational mechanistic data, which suggest that this intermediate should very readily and spontaneously convert to the epoxide species. The key question then becomes what is actually observed in the X-ray experiments. Here we report additional computational studies undertaken with the hope of clarifying this issue. The results obtained for active site models hosting both the native and the alternative (4-sulfonylcatechol) substrate indicate that the stability of the gem diol species is substantially increased if an electron and a proton are added. If this occurs somehow, the lifetime of the intermediate should be sufficient to observe it.     

Volatile metabolic profiles of cell suspension cultures of Lavandula vera,Nicotiana tabacum and Helianthus annuus,cultivated under different regimes

Cell suspension cultures of Lavandula vera (Lamiaceae), Nicotiana tabacum (Solanaceae), and Helianthus annuus (Asteraceae) were cultivated in three different ways: in shake flasks both as free suspensions and in two‐phase systems (in the presence of Amberlite XAD‐4 resin as a second phase), as well as in 3‐L stirred tank reactor, and their volatile metabolic profiles were studied using GC‐MS. A number of compounds, some of them having allelochemical and biological activities, were identified in all the three cell suspension cultures under study. Also the presence of some compounds, unusual for the intact plants, was observed. It was found that the cultivation mode strongly influences the production and the transport (secretion into the culture medium) of the low‐molecular‐mass volatile metabolites. Principal component analyses of 12 common hydrocarbons showed discrimination between the different cultivation modes (shake flasks and two‐phase systems cultivation) by first principal component (PC1) and second principal component (PC2).     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

Dead-end elimination with backbone flexibility

MOTIVATION: Dead-End Elimination (DEE) is a powerful algorithm capable of reducing the search space for structure-based protein design by a combinatorial factor. By using a fixed backbone template, a rotamer library, and a potential energy function, DEE identifies and prunes rotamer choices that are provably not part of the Global Minimum Energy Conformation (GMEC), effectively eliminating the majority of the conformations that must be subsequently enumerated to obtain the GMEC. Since a fixed-backbone model biases the algorithm predictions against protein sequences for which even small backbone movements may result in a significantly enhanced stability, the incorporation of backbone flexibility can improve the accuracy of the design predictions. If explicit backbone flexibility is incorporated into the model, however, the traditional DEE criteria can no longer guarantee that the flexible-backbone GMEC, the lowest-energy conformation when the backbone is allowed to flex, will not be pruned. RESULTS: We derive a novel DEE pruning criterion, flexible-backbone DEE (BD), that is provably accurate with backbone flexibility, guaranteeing that no rotamers belonging to the flexible-backbone GMEC are pruned; we also present further enhancements to BD for improved pruning efficiency. The results from applying our novel algorithms to redesign the beta1 domain of protein G and to switch the substrate specificity of the NRPS enzyme GrsA-PheA are then compared against the results from previous fixed-backbone DEE algorithms. We confirm experimentally that traditional-DEE is indeed not provably-accurate with backbone flexibility and that BD is capable of generating conformations with significantly lower energies, thus confirming the feasibility of our novel algorithms. AVAILABILITY: Contact authors for source code.     

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.