Near-infrared heavy-atom-modified fluorescent dyes for base-calling in DNA-sequencing applications using temporal discrimination

A series of near-IR fluorescent dyes were prepared which contained an intramolecular heavy atom for altering the fluorescence lifetimes to produce a set of probes appropriate for base-calling in a single-lane DNA sequencing format. The heavy-atom modification consisted of an intramolecular halogen situated on a remote section of the chromophore in order to minimize the perturbation on the lifetimes and fluorescence quantum yields. In addition, the dye series possessed an isothiocyanate functional group to allow facile attachment to sequencing primers. The unconjugated dyes showed similar absorption and emission maxima (lambda abs = 765-768 nm; lambda em = 794-798 nm) as well as fluorescence quantum yields that were invariant, within experimental error, with the heavy atom. However, the lifetimes of these dyes were found to vary with the identity of the halogen substitution (I, tau f = 947 ps; F, tau f = 843 ps, measured in methanol), with an average variation within the dye series of 35 ps. The spectroscopic properties of the free dyes and the dyes conjugated to sequencing primers on the 5'-end of the oligonucleotide were determined in a DNA-sequencing matrix (denaturing gels containing formamide). The results indicated slight differences in the fluorescence properties of the free dyes compared to those of the dye/ primer conjugates in this particular matrix. Inspection of the ground-state absorption spectra showed significant aggregation for the free dyes in this solution, but the conjugated dyes exhibited no sign of aggregation due to the highly anionic nature of the oligonucleotide. The fluorescence lifetimes of the dye/primer conjugates demonstrated lifetimes which ranged from 735 to 889 ps, with an average variation of 51 ps, an adequate difference to allow facile discrimination of these dyes in DNA-sequencing conditions. In addition, the free solution electrophoretic mobilities of the native heavy-atom-modified dyes were found to be very similar. When the dye/primer conjugates were electrophoresed in a cross-linked polyacrylamide gel electrophoresis capillary column, they comigrated, indicating that, in single-lane sequencing applications, when utilizing these dyes, no postrun corrections would be required to correct for dye-dependent mobility shifts.     

The traditional and emerging role of nuts in healthful diets

Throughout history, nuts have been a staple food providing energy, protein, essential fatty acids, vitamins, and minerals. Today, nuts are classified as part of the USDA Food Guide Pyramid's Meat/ Meat Alternate Group. Foods in this group contribute protein as well as important vitamins and minerals to the diet. Nuts are also being studied for their potential health benefits. Research suggests that there may be a connection between frequent nut consumption and a reduced incidence of coronary heart disease. Thus, tradition and promising scientific evidence combine to support the role of nuts in healthful eating.     

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit--identification of a soluble precursor of the small subunit in a hypB mutant

An active tryptic fragment of hydrogenase 2 from Escherichia coli has been isolated from the periplasmic face of the cytoplasmic membrane, and the large and small subunits N-terminally sequenced. The large subunit is encoded by the hybC gene and shows no N-terminal processing, other than removal of the initiator methionine during its biosynthesis. Both N-terminal and the subsequent internal tryptic-fragment amino acid sequence indicate that the small subunit is neither encoded by hybA, a gene previously identified as encoding the small subunit [Menon et al. (1994) J. Bacteriol. 176, 4416-4423], nor any of the remaining genes in the hyb operon. Genome sequence analysis revealed the presence of an open reading frame which could potentially encode the peptide sequences of the proteolysed small subunit. The gene, designated hyb0, lies directly upstream of, and is separated by two nucleotides from, the start of the hybA gene. Hyb0, which shares an approximate 40% identity with other hydrogenase small subunit amino acid sequences, is synthesised with an N-terminal signal sequence containing a twin-arginine motif which is probably required for export of the enzyme. In the mature enzyme the small subunit is proteolytically cleaved after Ala37. Immunological analysis of strains overproducing either recombinant Hyb0 or HybA using antibodies specific for hydrogenase 2, readily identified Hyb0 as the small subunit. In a pleiotropic hypB mutant, which is unable to insert nickel into the active site, both the large and small subunits accumulate as unprocessed, soluble forms, consistent with the two subunits being assembled and processed in a coordinated manner during biosynthesis.     

When hashes collide [applied cryptography]

Cryptography isn't in the public eye very often, but when it is, the news can be disconcerting. The authors discuss the issues that have arisen around the recently announced problems with the SHA-1 hash function and its ancestor, MD5.     

Effectiveness of the Woodruff School Doctoral Teaching Intern Program

This article discusses a unique teaching internship program that has been in place in the Woodruff School of Mechanical Engineering at Georgia Institute of Technology since 1990. The objective of the program is to provide doctoral students who are considering an academic career an opportunity to gain teaching experience under the tutelage of a faculty member. This article summarizes the major findings from student evaluations and from surveys of former student and faculty participants and discusses the apparent need and benefit of such a doctoral teaching intern program. Benefits and drawbacks of participation are discussed from the intern, faculty mentor, and student points of view. Conclusions are drawn about the overall effectiveness of the program and recommendations are made for implementing a successful teaching internship program.     

Plasticity of neuronal response properties in adult cat striate cortex

We have utilized an associative conditioning paradigm to induce changes in the receptive field (RF) properties of neurons in the adult cat striate cortex. During conditioning, the presentation of particular visual stimuli were repeatedly paired with the iontophoretic application of either GABA or glutamate to control postsynaptic firing rates. Similar paradigms have been used in kitten visual cortex to alter RF properties (Fregnac et al., 1988, 1992; Greuel et al., 1988; Shulz & Fregnac, 1992). Roughly half of the cells that were subjected to conditioning with stimuli differing in orientation were found to have orientation tuning curves that were significantly altered. In general, the modification in orientation tuning was not accompanied by a shift in preferred orientation, but rather, responsiveness to stimuli at or near the positively reinforced orientation was increased relative to controls, and responsiveness to stimuli at or near the negatively reinforced orientation was decreased relative to controls. A similar proportion of cells that were subjected to conditioning with stimuli differing in spatial phase were found to have spatial-phase tuning curves that were significantly modified. Conditioning stimuli typically differed by 90 deg in spatial phase, but modifications in spatial-phase angle were generally 30-40 deg. An interesting phenomenon we encountered was that during conditioning, cells often developed a modulated response to counterphased grating stimuli presented at the null spatial phase. We present an example of a simple cell for which the shift in preferred spatial phase measured with counterphased grating stimuli was comparable to the shift in spatial phase computed from a one-dimensional Gabor fit of the space-time RF profile. One of ten cells tested had a significant change in direction selectivity following associative conditioning. The specific and predictable modifications of RF properties induced by our associative conditioning procedure demonstrate the ability of mature visual cortical neurons to alter their integrative properties. Our results lend further support to models of synaptic plasticity where temporal correlations between presynaptic and postsynaptic activity levels control the efficiency of transmission at existing synapses, and to the idea that the mature visual cortex is, in some sense, dynamically organized.     

Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression

OBJECTIVE: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-specific cytokine, induces proliferation of endothelial cells and increases vascular permeability dramatically. All gliomas secrete significant amounts of VEGF, whereas meningiomas are variable in expression. Thus, we sought to determine whether the extent of VPF/VEGF expression in meningiomas correlated with differences in brain edema associated with these tumors. METHODS: Meningioma tissue samples from 37 patients (15 men, average age 65 +/- 13 yr; 22 women, average age 60 +/- 10 yr) who underwent surgery at or were referred to the University of Alabama Hospital were examined retrospectively for the extent of expression of immunoreactive VPF/VEGF. Additionally, peritumoral edema was assessed on a blinded basis radiographically from preoperative magnetic resonance imaging scans. Selected specimens were examined by in situ hybridization to document the source of VPF/VEGF. RESULTS: The predominant meningioma subclassifications were transitional (57%) or meningothelial (27%) subtypes. VPF/VEGF immunoreactivity ranged from 0 to 3.5, with a median value of 2 on a subjective 5-point scale; magnetic resonance imaging-assessed edema ranged in extent from 0 to 4 (subjective 5-point scale), with a median value of 2.5. The correlation of determination (R2) of magnetic resonance imaging-assessed tumor edema rating and VPF/VEGF staining intensity rating was 0.6087 (r = 0.78; P = 0.0001). In situ hybridization localized VPF/VEGF messenger ribonucleic acid in meningioma cells and not in normal parenchymal brain cells. CONCLUSION: These data suggest that meningioma-associated edema may be a result of the capacity of meningioma cells to produce VPF/VEGF locally, leading to increased tumor neovascularization and enhanced vascular permeability.