Integrated minimum-set primers and unique probe design algorithms for differential detection on symptom-related pathogens

Motivation: Differential detection on symptom-related pathogens(SRP) is critical for fast identification and accurate controlagainst epidemic diseases. Conventional polymerase chain reaction(PCR) requires a large number of unique primers to amplify selectedSRP target sequences. With multiple-use primers (mu-primers),multiple targets can be amplified and detected in one PCR experimentunder standard reaction condition and reduced detection complexity.However, the time complexity of designing mu-primers with thebest heuristic method available is too vast. We have formulatedminimum-set mu-primer design problem as a set covering problem(SCP), and used modified compact genetic algorithm (MCGA) tosolve this problem optimally and efficiently. We have also proposednew strategies of primer/probe design algorithm (PDA) on combiningboth minimum-set (MS) mu-primers and unique (UniQ) probes. Designedprimer/probe set by PDA-MS/UniQ can amplify multiple genes simultaneouslyupon physical presence with minimum-set mu-primer amplification(MMA) before intended differential detection with probes-arrayhybridization (PAH) on the selected target set of SRP. Results: The proposed PDA-MS/UniQ method pursues a much smallernumber of primers set compared with conventional PCR. In thesimulation experiment for amplifying 12 669 target sequences,the performance of our method with 68% reduction on requiredmu-primers number seems to be superior to the compared heuristicapproaches in both computation efficiency and reduction percentage.Our integrated PDA-MS/UniQ method is applied to the differentialdetection on 9 plant viruses from 4 genera with MMA and PAHof 11 mu-primers instead of 18 unique ones in conventional PCRwhile amplifying overall 9 target sequences. The results ofwet lab experiments with integrated MMA-PAH system have successfullyvalidated the specificity and sensitivity of the primers/probesdesigned with our integrated PDA-MS/UniQ method. Contact: cykao{at}csie.ntu.edu.tw Supplementary information: http://www.csie.ntu.edu.tw/~cykao/pda/     

Androgen receptor (AR) NH2- and COOH-terminal interactions result in the differential influences on the AR-mediated transactivation and cell growth

Early reports showed that androgen receptor (AR) NH2- and COOH-terminal (N-C) interaction was important for full AR function. However, the influence of these interactions on the AR in vivo effects remains unclear. Here we tested some AR-associated peptides and coregulators to determine their influences on AR N-C interaction, AR transactivation, and AR coregulator function. The results showed that AR coactivators such as ARA70N, gelsolin, ARA54, and SRC-1 can enhance AR transactivation but showed differential influences on the N-C interaction. In contrast, AR corepressors ARA67 and Rad9 can suppress AR transactivation, with ARA67 enhancing and Rad9 suppressing AR N-C interaction. Furthermore, liganded AR C terminus-associated peptides can block AR N-C interaction, but only selective peptides can block AR transactivation and coregulator function. We found all the tested peptides can suppress prostate cancer LNCaP cell growth at different levels in the presence of 5alpha-dihydrotestosterone, but only the tested FXXLF-containing peptides, not FXXMF-containing peptides, can suppress prostate cancer CWR22R cell growth. Together, these results suggest that the effects of AR N-C interactions may not always correlate with similar effects on AR-mediated transactivation and/or AR-mediated cell growth. Therefore, drugs designed by targeting AR N-C interaction as a therapeutic intervention for prostate cancer treatment may face unpredictable in vivo effects.     

Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by Mouse Pelle-like kinase/interleukin-1 receptor-associated kinase: cross-talk between inflammatory signaling and insulin signaling that may contribute to insulin resistance

Inflammation contributes to insulin resistance in diabetes and obesity. Mouse Pelle-like kinase (mPLK, homolog of human IL-1 receptor-associated kinase (IRAK)) participates in inflammatory signaling. We evaluated IRS-1 as a novel substrate for mPLK that may contribute to linking inflammation with insulin resistance. Wild-type mPLK, but not a kinase-inactive mutant (mPLK-KD), directly phosphorylated full-length IRS-1 in vitro. This in vitro phosphorylation was increased when mPLK was immunoprecipitated from tumor necrosis factor (TNF)-alpha-treated cells. In NIH-3T3(IR) cells, wild-type mPLK (but not mPLK-KD) co-immunoprecipitated with IRS-1. This association was increased by treatment of cells with TNF-alpha. Using mass spectrometry, we identified Ser(24) in the pleckstrin homology (PH) domain of IRS-1 as a specific phosphorylation site for mPLK. IRS-1 mutants S24D or S24E (mimicking phosphorylation at Ser(24)) had impaired ability to associate with insulin receptors resulting in diminished tyrosine phosphorylation of IRS-1 and impaired ability of IRS-1 to bind and activate PI-3 kinase in response to insulin. IRS-1-S24D also had an impaired ability to mediate insulin-stimulated translocation of GLUT4 in rat adipose cells. Importantly, endogenous mPLK/IRAK was activated in response to TNF-alpha or interleukin 1 treatment of primary adipose cells. In addition, using a phospho-specific antibody against IRS-1 phosphorylated at Ser(24), we found that interleukin-1 or TNF-alpha treatment of Fao cells stimulated increased phosphorylation of endogenous IRS-1 at Ser(24). We conclude that IRS-1 is a novel physiological substrate for mPLK. TNF-alpha-regulated phosphorylation at Ser(24) in the pleckstrin homology domain of IRS-1 by mPLK/IRAK represents an additional mechanism for cross-talk between inflammatory signaling and insulin signaling that may contribute to metabolic insulin resistance.     

Structural characterization of the [Pco/o(2)] compound of cytochrome c oxidase

The structural properties of a key transient oxygen intermediate of cytochrome c oxidase, P(R), remain an enigma, although inferences have been drawn from its equilibrium analogues, [Pco/o(2)] , P(H) and P(M). With resonance Raman spectroscopy, an oxygen isotope-sensitive band at 806 cm(-1) was observed in [Pco/o(2)] produced by adding CO and O(2) to the resting enzyme. The vibrational band shifted to 771 cm(-1) upon isotopic substitution of (16)O(2) with (18)O(2). The same modes at 806 and 771 cm(-1) were present simultaneously when the mixed isotope, (18)O(16)O, was employed, indicating that in [Pco/o(2)] the O-O bond is cleaved, resulting in a Fe(4+)O(2-) structure. This result unifies the nature of the three equilibrium analogues of the P(R) intermediate.     

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.     

A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation

Four small ubiquitin-related modifier (SUMO) genes have been identified in humans. However, little is known about the basic biology of SUMO-4. Here, we report that SUMO-4 differs from SUMO-1, -2, and -3 in that the maturation process of SUMO-4 to active form containing C-terminal di-glycine residues is inhibited by a unique proline residue located at position 90 (Pro-90). Although, both the hydrolase and isopeptidase activities of SUMO peptidases are significantly diminished by Pro-90 as compared to Gln-90 (glutamine) in mutated SUMO genes, only the defective hydrolase activity appears to be biologically relevant. Native SUMO-4, thus, appears to be unable to form covalent isopeptide bonds with substrates. A biological role of SUMO-4, through non-covalent interactions is proposed.     

Diffusion and electrophoretic mobility of single-stranded RNA from molecular dynamics simulations

Hydrodynamic properties of small single-stranded RNA homopolymers with three and six nucleotides in free solution are determined from molecular dynamics simulations in explicit solvent. We find that the electrophoretic mobility increases with increasing RNA length, consistent with experiment. Diffusion coefficients of RNA, corrected for finite-size effects and solvent viscosity, agree well with those estimated from experiments and hydrodynamic calculations. The diffusion coefficients and electrophoretic mobilities satisfy a Nernst-Einstein relation in which the effective charge of RNA is reduced by the charge of transiently bound counterions. Fluctuations in the counterion atmosphere are shown to enhance the diffusive spread of RNA molecules drifting along the direction of the external electric field. As a consequence, apparent diffusion coefficients measured by capillary zone electrophoresis can be significantly larger than the actual values at certain experimental conditions.