Virus Particles Monitored by Fluorescence Spectroscopy: A Potential Detection Assay for Macromolecular Assembly¶

Native fluorescence spectroscopy was used for in situ investigations of two lipid‐containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses φ6 and φ12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.     

Synthesis and chemistry of some pyridazine nucleosides related to certain 5-substituted pyrimidine nucleosides

Condensation of 3,4-dichloro-6-[(trimethylsilyl)oxy] pyridazine ( 3 ) with 1-O-acetyl-2,3,5-tri-O-benzoyl-β- D -ribofuranose ( 4 ), by the stannic chloride catalyzed procedure, has furnished 3,4-dichloro-1-(2,3,5-tri-O-benzoyl-β- D -ribofuranosyl) pyridazin-6-one ( 5 ). Nucleophilic displacement of the chloro groups and removal of the benzoyl blocking groups from 5 has furnished 3-chloro-4-methoxy-, 3,4-dimethoxy-, 4-amino-3-chloro-, 3-chloro-4-methylamino-, 3-chloro-4-hydroxy-, and 4-hydroxy-3-methoxy-1-β- D -ribofuranosylpyridazin-6-one. An unusual reaction of 5 with dimethylamine is reported. Condensation of 4,5-dichloro-3-nitro-6-[(trimethylsilyl)oxy]pyridazine with 4 yielded 4,5-dichloro-3-nitro-1-(2,3,5-tri-O-benzoyl-β- D -ribofuranosyl)pyridazin-6-one ( 24 ). Nucleophilic displacement of the aromatic nitro groups from 24 is discussed. Condensation of 3 with 3,5-di-O-p-toluoyl 2-deoxy- D -erythro-pentofuranosyl chloride ( 28 ) afforded an α, β mixture of 2-deoxy nucleosides. The synthesis of certain 3-substituted pyridazine 2′-deoxy necleosides are reported.     

Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

In situ tissue pathology from spatially encoded mass spectrometry classifiers visualized in real time through augmented reality

Integration between a hand-held mass spectrometry desorption probe based on picosecond infrared laser technology (PIRL-MS) and an optical surgical tracking system demonstrates in situ tissue pathology from point-sampled mass spectrometry data. Spatially encoded pathology classifications are displayed at the site of laser sampling as color-coded pixels in an augmented reality video feed of the surgical field of view. This is enabled by two-way communication between surgical navigation and mass spectrometry data analysis platforms through a custom-built interface. Performance of the system was evaluated using murine models of human cancers sampled in situ in the presence of body fluids with a technical pixel error of 1.0 ± 0.2 mm, suggesting a 84% or 92% (excluding one outlier) cancer type classification rate across different molecular models that distinguish cell-lines of each class of breast, brain, head and neck murine models. Further, through end-point immunohistochemical staining for DNA damage, cell death and neuronal viability, spatially encoded PIRL-MS sampling is shown to produce classifiable mass spectral data from living murine brain tissue, with levels of neuronal damage that are comparable to those induced by a surgical scalpel. This highlights the potential of spatially encoded PIRL-MS analysis for in vivo use during neurosurgical applications of cancer type determination or point-sampling in vivo tissue during tumor bed examination to assess cancer removal. The interface developed herein for the analysis and the display of spatially encoded PIRL-MS data can be adapted to other hand-held mass spectrometry analysis probes currently available.

Integration between a hand-held mass spectrometry desorption probe based on picosecond infrared laser technology (PIRL-MS) and an optical surgical tracking system demonstrates in situ tissue pathology from point-sampled mass spectrometry data.     

A thermochemical analysis of inhalational anesthetics

The mechanism of the anesthetic process is of interest both to the clinician and to the pharmacologist. However, this is still an unsettled issue and a multitude of models have been proposed for the process. Noticing that most models propose either a molecular perturbation by the agents or an effect on some colligative property, we explore in this article the thermodynamical consequences of these postulations. Comparison of these with experimental findings is then made. The comparison shows the inconsistency of many of the models with the facts: (i) it refutes the long accepted conviction, culminated in the unitary hypothesis, that general anesthetics act not at a particular receptor site but invariably on all. Some consequences of this finding are demonstrated. (ii) it implies that a simple phospholipid medium is not feasible as an anesthetic site. (iii) it infers that proteins do have the properties required from anesthetic sites.Dedicated to Prof. Menachem Steinberg on the occasion of his 65th birthday     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Hybrid electron cyclotron resonance-radio-frequency plasma etching of III–V semiconductors in Cl2-based discharges. Part I: GaAs and related compounds

A systematic study has been performed of the dry etching characteristics of GaAs, Al_0.3Ga_0.7As, and GaSb in chlorine-based electron cyclotron resonance (ECR) discharges. The gas mixtures investigated were CCl₂F₂/O₂, CHCl₂F/O₂, and PCl₃. The etching rates of all three materials increase rapidly with applied RF power, while the addition of the microwave power at moderate levels (150 W) increases the etch rates by 20–80%. In the microwave discharges, the etch rates decrease with increasing pressure, but at 1 m Torr it is possible to obtain usable rates for self-bias voltages 100 V. Of the Freon-based mixtures, CHCl₂F provides the least degradation of optical (photoluminescence) and electrical (diode ideality factors and Schottky barrier heights) properties of GaAs as a result of dry etching. Smooth surface morphologies are obtained on all three materials provided the microwave power is limited to 200 W. Above this power, there is surface roughening evident with all of the gas mixtures investigated.     

Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.     

Pyramids in the complex projective plane

We study the critical points of the diameter functional on the n-fold Cartesian product of the complex projective plane C P ² with the Fubini-Study metric. Such critical points arise in the calculation of a metric invariant called the filling radius, and are akin to the critical points of the distance function. We study a special family of such critical points, P _kC P ¹C P ², k=1,2... We show that P _k is a local minimum of by verifying the positivity of the Hessian of (a smooth approximation to) at P _k. For this purpose, we use Shirokov's law of cosines and the holonomy of the normal bundle of C P ¹C P ². We also exhibit a critical point of , given by a subset which is not contained in any totally geodesic submanifold of C P ².