The Plasticity of the Carbohydrate Recognition Domain Dictates the Exquisite Mechanism of Binding of Human Macrophage Galactose-Type Lectin

The human macrophage galactose-type lectin (MGL), expressed on macrophages and dendritic cells (DCs), modulates distinct immune cell responses by recognizing N-acetylgalactosamine (GalNAc) containing structures present on pathogens, self-glycoproteins, and tumor cells. Herein, NMR spectroscopy and molecular dynamics (MD) simulations were used to investigate the structural preferences of MGL against different GalNAc-containing structures derived from the blood group A antigen, the Forssman antigen, and the GM2 glycolipid. NMR spectroscopic analysis of the MGL carbohydrate recognition domain (MGL-CRD, C181-H316) in the absence and presence of methyl α-GalNAc (α-MeGalNAc), a simple monosaccharide, shows that the MGL-CRD is highly dynamic and its structure is strongly altered upon ligand binding. This plasticity of the MGL-CRD structure explains the ability of MGL to accommodate different GalNAc-containing molecules. However, key differences are observed in the recognition process depending on whether the GalNAc is part of the blood group A antigen, the Forssman antigen, or GM2-derived structures. These results are in accordance with molecular dynamics simulations that suggest the existence of a distinct MGL binding mechanism depending on the context of GalNAc moiety presentation. These results afford new perspectives for the rational design of GalNAc modifications that fine tune MGL immune responses in distinct biological contexts, especially in malignancy.     

Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome [P(r) and P(fr) forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.     

Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 × HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.     

Zn2+-dependent peptide nucleic acids probes

Binding of bis-picolylamine-naphthalene diimide-peptide nucleic acid conjugates to complementary DNA is strongly dependent upon Zn2+; ultimately, hybridization is switched ON in the presence muM Zn2+.     

Computational studies of carbon nanotube-hydrocarbon bond strengths at nanotube ends: effect of link heteroatom and hydrocarbon structure

Semiempirical and density functional electronic structure theory methods were used to study SWNT-X--R bond strengths, where the single-walled carbon nanotube (SWNT) had an armchair or zigzag structure, the link heteroatom X was O, N(H), or S and the hydrocarbon chain R was CH(2)CH(3), CH(OH)CH(3), CHCH(2), or CH(CF(3))CH(3). In all systems the hydrocarbon was bonded to the end of the nanotube. The SWNT-X--R bond (that is, the bond joining the link atom to the hydrocarbon) is more than 0.4 eV stronger for armchair than for zigzag nanotubes with the same diameters, irrespective of whether O, N, or S are used as link atoms or whether OH, C==C, or CF(3) groups are present in the hydrocarbon chain. This raises the possibility for selective manipulation of armchair/zigzag nanotubes using a variety of link atoms and hydrocarbon structures. The SWNT-O--CH(CF(3))CH(3) bond is weaker than the SWNT-O--CH(2)CH(3) bond (for both armchair and zigzag nanotubes), while inclusion of a double bond in the ethyl chain increases the bond strengths. Also, SWNT-S--CH(2)CH(3) and SWNT-N(H)--CH(2)CH(3) bonds are stronger than SWNT-O--CH(2)CH(3) bonds.     

Measurements of side-chain 13C-13C residual dipolar couplings in uniformly deuterated proteins

13C-only spectroscopy was used to measure multiple residual (13)C-(13)C dipolar couplings (RDCs) in uniformly deuterated and (13)C-labeled proteins. We demonstrate that (13)C-start and (13)C-observe spectra can be routinely used to measure an extensive set of the side-chain residual (13)C-(13)C dipolar couplings upon partial alignment of human ubiquitin in the presence of bacteriophages Pf1. We establish that, among different broadband polarization transfer schemes, the FLOPSY family can be used to exchange magnetization between a J coupled network of spins while largely decoupling dipolar interactions between these spins. An excellent correlation between measured RDCs and the 3D structure of the protein was observed, indicating a potential use of the (13)C-(13)C RDCs in the structure determination of perdeuterated proteins.     

Droplet microfluidics for amplification-free genetic detection of single cells

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.     

CRDOCK: An Ultrafast Multipurpose Protein-Ligand Docking Tool

An ultrafast docking and virtual screening program, CRDOCK, is presented that contains (1) a search engine that can use a variety of sampling methods and an initial energy evaluation function, (2) several energy minimization algorithms for fine tuning the binding poses, and (3) different scoring functions. This modularity ensures the easy configuration of custom-made protocols that can be optimized depending on the problem in hand. CRDOCK employs a precomputed library of ligand conformations that are initially generated from one-dimensional SMILES strings. Testing CRDOCK on two widely used benchmarks, the ASTEX diverse set and the Directory of Useful Decoys, yielded a success rate of ～75% in pose prediction and an average AUC of 0.66. A typical ligand can be docked, on average, in just ～13 s. Extension to a representative group of pharmacologically relevant G protein-coupled receptors that have been recently cocrystallized with some selective ligands allowed us to demonstrate the utility of this tool and also highlight some current limitations. CRDOCK is now included within VSDMIP, our integrated platform for drug discovery.     

Microfluidic platform for on-demand generation of spatially indexed combinatorial droplets

We propose a highly versatile and programmable nanolitre droplet-based platform that accepts an unlimited number of sample plugs from a multi-well plate, performs digitization of these sample plugs into smaller daughter droplets and subsequent synchronization-free, robust injection of multiple reagents into the sample daughter droplets on-demand. This platform combines excellent control of valve-based microfluidics with the high-throughput capability of droplet microfluidics. We demonstrate the functioning of a proof-of-concept device which generates combinatorial mixture droplets from a linear array of sample plugs and four different reagents, using food dyes to mimic samples and reagents. Generation of a one dimensional array of the combinatorial mixture droplets on the device leads to automatic spatial indexing of these droplets, precluding the need to include a barcode in each droplet to identify its contents. We expect this platform to further expand the range of applications of droplet microfluidics to include applications requiring a high degree of multiplexing as well as high throughput analysis of multiple samples.     

Uncertainty estimation to evaluate mass balances on a combustion system

Mass balances of ash and potassium for a fluidized bed combustor were performed incorporating measurement uncertainties. The total output mass of ash or a chemical element should be equal to the mass in the input fuel; however, this is not often achieved. A realistic estimation of recovery uncertainty can support the reliability of a mass balance. Estimation of uncertainty helps to establish a reliable evaluation of the recovery ratio of ash mass and elemental mass. This may clarify whether any apparent lack in closing the mass balance can be attributed to uncertainties. The evaluation of measurement uncertainty for different matrices, namely coal, biomass, sand and ashes from different streams was based on internal quality control data and external quality control data, namely analysis of samples from proficiency tests or use of a certified reference material. The evaluation of intermediate precision and trueness allowed the estimation of measurement uncertainty. Due to the different physic and chemical characteristics of the studied matrices, the uncertainty of precision was evaluated using R-charts of data obtained from the analysis of duplicates for the majority of samples. This allowed evaluating sample heterogeneity effects. The instrumental acceptance criterion was also considered and included in the combined uncertainty. The trueness was evaluated using data from several proficiency tests and from analysis of a certified reference material or sample spiking. Statistically significant bias was included.