A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles

Using a fluorescence-based method, we have determined the number of thiol-derivatized single-stranded oligonucleotides bound to gold nanoparticles and their extent of hybridization with complementary oligonucleotides in solution. Oligonucleotide surface coverages of hexanethiol 12-mer oligonucleotides on gold nanoparticles (34 +/- 1 pmol/cm2) were significantly higher than on planar gold thin films (18 +/- 3 pmol/cm2), while the percentage of hybridizable strands on the gold nanoparticles (1.3 +/- 0.3 pmol/cm2, 4%) was lower than for gold thin films (6 +/- 2 pmol/cm2, 33%). A gradual increase in electrolyte concentration over the course of oligonucleotide deposition significantly increases surface coverage and consequently particle stability. In addition, oligonucleotide spacer sequences improve the hybridization efficiency of oligonucleotide-modified nanoparticles from approximately 4 to 44%. The surface coverage of recognition strands can be tailored using coadsorbed diluent oligonucleotides. This provides a means of indirectly controlling the average number of hybridized strands per nanoparticle. The work presented here has important implications with regard to understanding interactions between modified oligonucleotides and metal nanoparticles, as well as optimizing the sensitivity of gold nanoparticle-based oligonucleotide detection methods.     

Sampling and analysis of particulate matter by glow discharge atomic emission and mass spectrometries

The direct introduction of particulate matter into glow discharge atomic emission and mass spectrometry sources through a particle beam/momentum separator apparatus is described. Vacuum action through a narrow (0.0625 in. i.d.) stainless steel tube allows the introduction of discrete samples of NIST SRM 1648 urban particulate matter (UPM) and caffeine in powder form. Introduction of "ambient" airborne particulate matter is also possible. Particles passing through the aerodynamic momentum separator impinge on the heated (～200-250 °C) inner surface of the glow discharge plasma volume and are flash-vaporized. The resultant atoms/molecules are subjected to excitation/ionization collisions within the low-pressure (0.5-5 Torr of He or Ar) plasma, producing characteristic photon emission and/or signature ionic species. In this way, atomic emission and mass spectrometry identification of particle constituents is possible. Basic design aspects of the apparatus are presented, and demonstrations of atomic emission detection of the constituents in the NIST SRM illustrate the general characteristics of the approach. Transient atomic emission signals are captured for the introduction of preweighed, discrete samples, with the integrated areas used to construct analytical response curves. Limits of detection using this relatively simple atomic emission system are on the order of tens of nanograms for sample masses of ～50 μg. Mass spectrometric monitoring of introduced caffeine particles and a mixture of polycyclic aromatic hydrocarbons (PAHs) illustrates the ability of the glow discharge plasma to produce high-quality, library (electron impact) searchable mass spectra of molecular species while also yielding isotopic identification of elemental components of the UPM. Limits of detection for Fe in the NIST SRM are on the order of 175 ng of material, equivalent to ～7 ng of analyte Fe. It is believed that the small size, low power consumption, ease of operation, and multimode sampling capabilities (AES/MS) of the particle beam-glow discharge (PB-GD) apparatus hold promise for applications in continuous monitoring and discrete particle sampling.     

In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.     

Residual analysis in random regressions using SAS and S-PLUS

A program package RRAP: Random Regression Residual Analysis Program using SAS [1] and S-PLUS [2] is available for performing random regression residual analysis. The PROCEDURE MIXED from SAS is used for statistical inference. Both elementary-level and individual-level residuals are used. The S-PLUS programs provide: (1) a transformation to orthogonalize the elementary-level correlated residuals for standard regression residual analyses; and (2) several statistics and plots for checking model assumptions, assessing model fitting and detecting outlying individuals. RRRAP starts with a SAS Macro RRRAPMAC on the data followed by a S-PLUS Program DoRRRAP on a UNIX system.     

CAN SIZE EXCLUSION CHROMATOGRAPHY WITH ELEMENT SPECIFIC DETECTION THE D 2007-80 WITH ASPHALTENE PRECIPITATION (SARA)SEPARATION AND HYDROGEN DISTRIBUTION BY NMR HELP AT ALL IN PREDICTING RESIDUUM PROCESSABILITY?

Over the years, several analytical methods have been applied to various heavy crude oil residua and their processed products to understand the chemistry behind residuum upgrading processes. The ultimate aim has been to predict prccessability of specific feeds. However, few, if any analytical methods have been found which adequately perform this task. This paper examines selected processing experiments by the following techniques - clusion chromatography with element specific detection, D 2007-80 with as-phaltene precipitation (SARA) separation, hydrogen distribution and incorporation by NMR - cusses whether the analytical technique has any potential to predict prccessability

From the size exclusion chromatography with element specific detection studies, an intimate relationship appears between the catalyst pore size and molecule size based on examination of the size behavior of feeds and pilot-plant products. From the D 2007-80 and asphaltene separations, the quality of asphaltenes appears to be related to the relative ease of processability of at least two different feeds. From the hydrogen distribution studies, hydrogen utilization was found to be feed dependent and could be directed by processing type. All these trends have some potential towards the formulation of a residuum processability scheme, however no method is as yet globally satisfactory.     

Wind-induced accidents of road vehicles

This paper describes the results of a project that collected and collated data for windinduced vehicle accidents that occurred in the United Kingdom during the major storm of 25 January 1990. The results of this data analysis are used to determine which vehicles are most at risk during windy periods, the nature of the accidents that were caused, and the likely values of accident wind speeds. Also the adequacy of a computer program that can predict such wind speeds (the program BLOWOVER) is assessed as far as the available data allows.     

Do routine antibiotics after loop diathermy excision reduce morbidity?

AIMS: To determine functional results after unilateral and bilateral cataract surgery in children with different aphakic optical correction. METHODS: In this retrospective study, we evaluated visual acuity and binocular vision in 107 children who underwent cataract surgery during the 10 year period from 1985 to 1995. Aphakia was corrected by an intracapsular intraocular lens (IOL), spectacles or contact lenses. RESULTS: Mean visual acuity was > 20/40 (< 0.3 log MAR) with normal binocular vision in 58 children over 7 months of age operated on for bilateral cataracts. Pseudophakic eyes regained visual acuity > 20/63 (< 0.5 log MAR) more often (90%) than aphakic eyes (46%) (p < 0.001). Binocular vision was also achieved more often after IOL implantation (p < 0.001). Visual outcome of early bilateral cataracts was less satisfactory in children with abnormal foveolar function. For 49 children who had surgery for unilateral cataracts, prognosis was poor when surgery was performed before the age of 7 months. For cataract surgery in older children (> or = 7 months) mean visual acuities were better with IOL implantation (p < 0.05). CONCLUSION: Cataract surgery with unilateral and bilateral IOL implantation can provide a beneficial effect on final visual outcome in children who are operated on before abnormal foveolar function develops.     

Local rules simulation of the kinetics of virus capsid self-assembly

Jurkat T cells undergo rapid apoptosis upon stimulation of the Fas/APO-1 (CD95) receptor. We examined the role of the mitogen-activated protein kinase (MAPK) cascade as a negative regulator of Fas-mediated apoptosis. To this end, we used both physiologic and artificial activators of MAPK, all of which activate MAPK by distinct routes. MAPK activity could be efficiently elevated by two T cell mitogens, the lectin PHA and an agonistic Ab to the T cell receptor complex as well as by the type 1 and 2A phosphatase inhibitor, calyculin A, and the protein kinase C-activating phorbol ester, tetradecanoyl phorbol acetate. All these treatments were effective in preventing the characteristic early and late features of Fas-mediated apoptosis, including activation of caspases. Our results indicate that the elevated MAPK activities intervene upstream of caspase activation. The degree of MAPK activation by the different stimuli used in our study corresponds well to their potency to inhibit apoptosis, indicating that MAPK activation serves as an efficient modulator of Fas-mediated apoptosis. The role of MAPK in modulation of Fas-mediated apoptosis was further corroborated by transient transfection with constitutively active MAPK kinase, resulting in complete inhibition of the Fas response, whereas transfection with a dominant negative form of MAPK kinase had no effect. Furthermore, the apoptosis inhibitory effect of the MAPK activators could be abolished by the specific MAPK kinase inhibitor PD 098059. Modulation of Fas responses by MAPK signaling may determine the persistence of an immune response and may explain the insensitivity of recently activated T cells to Fas receptor stimulation.