An integrated approach for automating validation of extracted ion chromatographic peaks

SUMMARY: Accurate determination of extracted ion chromatographic peak areas in isotope-labeled quantitative proteomics is difficult to automate. Manual validation of identified peaks is typically required. We have integrated a peak confidence scoring algorithm into existing tools which are compatible with analysis pipelines based on the standards from the Institute for Systems Biology. This algorithm automatically excludes incorrectly identified peaks, improving the accuracy of the final protein expression ratio calculation. SOURCE AND SUPPLEMENTARY INFORMATION: http://www.chem.uky.edu/research/lynn/Nelson.pdf.     

A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.     

Effect of an exotic herbivore,Adelges tsugae,on photosynthesis of a highly susceptible Tsuga host,with notes on conspecifics

Hemlocks are significant components of temperate forests of Asia and North America, and in eastern North America, they are threatened by an exotic herbivore, the hemlock woolly adelgid, Adelges tsugae. The adelgid is native to Asia and northwestern North America, but is highly invasive in eastern North America where natural enemies are unable to regulate populations and eastern hemlock, Tsuga canadensis, is highly susceptible. In order to gain a better understanding of the metabolic effects of A. tsugae on eastern hemlock, we evaluated its effects on photosynthesis and also evaluated photosynthesis on Tsuga species from various geographic origins. We measured light-saturated photosynthesis (A _sat) and dark respiration of T. canadensis that were infested with adelgid and found a significant decrease in A _sat and a small but significant increase in dark respiration, suggesting that A. tsugae triggers a physiological response in eastern hemlock by decreasing metabolic activity. In a separate experiment, we also measured A _sat of five different hemlock species, including eastern hemlock, the Pacific Northwestern T. heterophylla and T. mertensiana, and the Asian T. diversifolia and T. chinensis. Only weakly significant differences in A _sat were found, with the highest rate in the eastern North American T. canadensis and the lowest in the Pacific Northwestern T. mertensiana. The relatively high photosynthetic rate of T. canadensis could possibly play a role in its susceptibility to A. tsugae. A better understanding of this metabolic response could help develop effective management strategies for combating the highly invasive A. tsugae.     

The DNA Replication Licensing Factor Miniature Chromosome Maintenance 7 Is Essential for RNA Splicing of Epidermal Growth Factor Receptor,c-Met,and Platelet-derived Growth Factor Receptor

Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221–248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.     

Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.     

Hemoglobin regulates the migration of glioma cells along poly(ε‐caprolactone)‐aligned nanofibers

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O₂)‐carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε‐caprolactone) (PCL)‐aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O₂ tensions ranging from 1 to 19% O₂. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL‐aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O₂ levels. The control data suggests that decreasing the O₂ tension in the incubator from 5 to 1% O₂ decreased aggregate dispersion on the PCL‐aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂, Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb‐based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids on nanofibers. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214–1220, 2014     

Production of fumonisins by Fusarium moniliforme strains from various substrates and geographic areas.

Strains of Fusarium moniliforme from different geographic areas and from corn and other substrates were tested for the ability to produce fumonisins in culture. The test results indicate that the potential exists for production of fumonisins by such strains in agricultural commodities and other substrates in widespread geographic areas.     

Amplification of a Cryptosporidium parvum gene fragment encoding thymidylate synthase.

Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.