Algal Flocculation with Synthetic Organic Polyelectrolytes

The feasibility of removing algae from water and wastewater by chemical flocculation techniques was investigated. Mixed cultures of algae were obtained from both continuous- and batch-fed laboratory reactors. Representative cationic, anionic, and nonionic synthetic organic polyelectrolytes were used as flocculants. Under the experimental conditions, chemically induced algal flocculation occurred with the addition of cationic polyelectrolyte, but not with anionic or nonionic polymers, although attachment of all polyelectrolyte species to the algal surface is shown. The mechanism of chemically induced algal flocculation is interpreted in terms of bridging phenomena between the discrete algal cells and the linearly extended polymer chains, forming a three-dimensional matrix that is capable of subsiding under quiescent conditions. The degree of flocculation is shown to be a direct function of the extent of polymer coverage of the active sites on the algal surface, although to induce flocculation by this method requires that the algal surface charge must concurrently be reduced to a level at which the extended polymers can bridge the minimal distance of separation imposed by electrostatic repulsion. The influence of pH, algal concentration, and algal growth phase on the requisite cationic flocculant dose is also reported.     

Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.Human cytomegalovirus (HCMV) is a widespread betaherpesvirus that causes lifelong persistent infections with occasional reactivations. While HCMV infection is usually clinically unapparent in the immunocompetent host, it can cause severe disseminated infections under conditions of immunosuppression, with manifestations in the lung, retina, and gastrointestinal tract, among others (12). Various cell types support viral replication, including epithelial cells and endothelial cells (ECs), smooth muscle cells, fibroblasts, and cells of hematopoietic origin (13, 14, 18, 19, 25, 26, 37). Among these target cells, endothelial cells are assumed to contribute particularly to hematogenous dissemination of HCMV (24).While recent clinical HCMV isolates are characterized by this broad cell tropism, the target cell range becomes restricted during long-term propagation on fibroblasts (28, 33). The underlying mechanism for this cell culture adaptation is a modulation within the viral genes UL128, UL130, and UL131A (8, 11). These three genes have been shown to be essential for infection of granulocytes, dendritic cells, epithelial cells, and endothelial cells but are dispensable for infection of fibroblasts (1, 9, 11, 34, 35). The encoded proteins pUL128, pUL130, and pUL131A were reported to form a complex with the viral glycoproteins gH and gL that is distinct from the glycoprotein complex gCIII (gH/gL/gO) (35). Whereas poorly endotheliotropic HCMV strains bear just the gH/gL/gO complex in their envelopes, highly endotheliotropic strains bear both gCIII variants: gH/gL/gO and gH/gL/pUL128-131A. Deletion of any of the three genes UL128-131A results in loss of EC tropism (11), most likely because only a complete complex of gH/gL and pUL128, pUL130, and pUL131A can efficiently function in endocytic entry in ECs (21). However, functional sites within the proteins (e.g., mediating binding to the viral complex partners or interaction with a putative cellular receptor) have not yet been identified. One approach to search for candidate protein-protein interaction sites is charge-cluster-to-alanine (CCTA) mutagenesis. This method is based on the assumption that clusters of charged amino acids tend to be exposed in the tertiary structure of a protein and are thus likely to be sites of interaction with other proteins. Replacement of these charged amino acids by uncharged alanines should then target protein-protein interaction sites without destroying the protein backbone (5, 7). Applying this method to HCMV pUL128, we were able to identify a central core region within pUL128 essential for EC infection as well as contributing sites in the N-terminal half and the C terminus of the protein (22). We now aimed to extend the study to the scanning of UL130 by markerless mutagenesis in the context of a highly endotheliotropic HCMV BACmid, TB40-BAC4. The resulting mutant viruses were then characterized with regard to their ability to infect ECs to identify the relevant parts of the protein.With regard to the role of UL130 in EC infection by endocytosis, the C-terminal part of pUL130 was of special interest. A frameshift mutation that changes the last 11 amino acids (aa) of pUL130 is the most prominent difference between the poorly endotheliotropic HCMV strain Towne and the highly endotheliotropic strain HCMV-TB40-BAC4 in this region (8, 11, 27). Rhee and Davis have described a cell-penetrating pentapeptide (CPP) motif (PFVYLI) mediating internalization by endocytosis, which is clathrin and caveolin independent but may involve lipid rafts (17). Not only do the last five amino acids of pUL130 (PNLIV) bear a striking similarity to this motif, but also the entry of HCMV into ECs has been reported to occur by an endocytic pathway (20, 23). Thus, we hypothesized that the pentapeptide motif PNLIV in pUL130 might be involved in mediating endocytic uptake of HCMV in ECs, and if so, deletion of this motif should result in a nonendotheliotropic virus. A number of CPPs that are thought to be taken up by endocytosis have now been described, including VPMLK, PMLKE, VPTLK, KLPVM, and others (32). These CPPs all bear some similarity, but the exact amino acid sequence seems to be irrelevant. We thus hypothesized for UL130 that a scrambled mutant (PNLIV changed to PINVL) should still be able to mediate endocytosis of HCMV in ECs. To test these assumptions we generated a series of mutant viruses where the PNLIV motif was either deleted, scrambled (PNLIV changed to PINVL), or exchanged against a known CPP (PFVYLI [17]) and characterized them with regard to EC infectivity.     

Impedance spectroscopy of alpha-beta tubulin heterodimer suspensions

Impedance spectroscopy is a technique that reveals information, such as macromolecular charges and related properties about protein suspensions and other materials. Here we report on impedance measurements over the frequency range of 1 Hz to 1 MHz of alpha-beta tubulin heterodimers suspended in a buffer. These and other polyelectrolyte suspensions show enormous dielectric responses at low frequencies, due both to the motion of charges suspended in the medium and to an electrical double layer that forms at each electrode-medium interface. We propose an equivalent circuit model to minimize electrode polarization effects and extract the intrinsic response of the bulk medium. At megaHertz frequencies, the conductivity increases with concentration below the critical concentration of approximately 1 mg/ml for microtubule polymerization, above which the conductivity decreases. This suggests that such measurements can be used to monitor the dynamics of microtubule polymerization. Finally, we obtain the net charge number per tubulin dimer of /Z/ = 306 in the saline buffer, which, if maintained as the dimers polymerized, would yield a linear charge density of 3.8 e/angstroms for the assembled microtubules. These results are potentially important for fundamental electrostatic processes in biomolecules and suggest the possibility of developing future bioelectronic applications.     

Tubulin dipole moment, dielectric constant and quantum behavior: computer simulations, experimental results and suggestions

We used computer simulation to calculate the electric dipole moments of the alpha- and beta-tubulin monomers and dimer and found those to be |p(alpha)| = 552D, |p(beta)| = 1193D and |p(alphabeta)| = 1740D, respectively. Independent surface plasmon resonance (SPR) and refractometry measurements of the high-frequency dielectric constant and polarizability strongly corroborated our previous SPR-derived results, giving Deltan/Deltac approximately 1.800 x 10(-3)ml/mg. The refractive index of tubulin was measured to be n(tub) approximately 2.90 and the high-frequency tubulin dielectric constant k(tub) approximately 8.41, while the high-frequency polarizability was found to be alpha(tub) approximately 2.1 x 10(-33)C m(2)/V. Methods for the experimental determination of the low-frequency p are explored, as well as ways to test the often conjectured quantum coherence and entanglement properties of tubulin. Biobits, bioqubits and other applications to bioelectronics are discussed.     

Morphological and membrane characteristics of spider and spindle cells isolated from rabbit sinus node

This study reports the comparative quantitative, morphological, and electrophysiological properties of two pacemaker cell types, spider and spindle-shaped cells, isolated from the rabbit sinoatrial node. Isolated nodal cells were studied with perforated and ruptured patch whole cell recording techniques. The basic spontaneous cycle length of the spider cells was 381 +/- 12 ms, and the basic spontaneous cycle length of the spindle cells was 456 +/- 17 ms (n = 12, P < 0.05). The spider cells had a more positive maximum diastolic potential (-54 +/- 1 mV) compared with the spindle cells (-68 +/- 1mV, P < 0.05). The overshoot and action potential amplitudes were also smaller in the spider cells. The hyperpolarization-activated inward (I(f)) current density, measured from their tail currents, was 15 +/- 1.3 pA/pF for the spider cells and 9 +/- 0.7 pA/pF for the spindle cells (P < 0.01). I(f) current activation voltage was more positive in the spider cells than the spindle cells. Isoproterenol (1 microM) decreased the spontaneous cycle length of the spider cells by 28 +/- 3% and the spindle cells by 20 +/- 1.5% (P < 0.05). Acetylcholine (0.5 microM) hyperpolarized the membrane potential of the spider cells to -86 +/- 0.7 mV and the spindle cells to -76 +/- 0.8 mV (P < 0.05). In summary, there are at least two distinct pacemaker cell types in the sinus node with different electrophysiological characteristics.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

Electro-optical behaviour of CuFe2O4@rGO nanocomposite for nonenzymatic detection of uric acid via the electrochemical method

Uric acid (UA) is a blood and urine component obtained as a metabolic by-product of purine nucleotides. Abnormalities in UA metabolism cause crystal deposition as monosodium urate and lead to various diseases such as gout, hyperuricemia, Lesch–Nyhan syndrome, etc. Monitoring these diseases requires a rapid, sensitive, selective, and portable detection approach. Therefore, this study demonstrates the hydrothermal synthesis of CuFe₂O₄/reduced graphene oxide (rGO) nanocomposite for selective detection of UA. After the nanocomposite synthesis, characterization was performed by X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, UV–visible spectrometry, atomic force spectroscopy, scanning electron microscopy, and electrochemical analysis. Furthermore, from the electrochemical analysis using cyclic voltammetry (CV), kinetic studies were carried out by varying the scan rate to obtain the diffusion coefficient, surface concentration, and rate of charge transfer to achieve a calibration curve that indicates the quasi reversible nature of the fabricated electrode with a linear regression coefficient of oxidation (R²: 0.9992) and reduction (R²: 0.9971) peaks. Moreover, the fabricated nonenzymatic amperometric sensor to detect UA with a linearity (R²: 0.9989) of 1–400 μM was highly sensitive (2.75 × 10⁻⁴ mAμM⁻¹ cm⁻²) and had a lower limit of detection (0.01231 μM) at pH 7.5 in phosphate-buffered saline solution. Therefore, the CuFe₂O₄/rGO/ITO-based nonenzymatic sensor could detect interfering agents and spiked real bovine serum samples with higher sensitivity and selectivity for UA detection.     

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org