Prospects of anionic nanolipoplexes in nanotherapy: transmission electron microscopy and light scattering studies

Currently nanosystems composed of polynucleotides and lipid vesicles (nanolipoplexes) are considered to be promising tools for gene therapeutics. Successful in vivo application of these vectors depends on their physicochemical, technological and biological characteristics including morphology, size distribution, molecular interactions and stability. Anionic nanoliposomes (DPPC:DCP:CHOL) were prepared by two different techniques, namely the conventional thin-film hydration method followed by extrusion, and the heating method (HM), in which no volatile solvent or detergent is used. A non-viral and non-cationic gene transfer vector was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) to the HM-nanoliposomes by the electrostatic mediation of Ca²⁺ ions. Transfection efficiency of the nanolipoplexes was evaluated using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Particle characterisation, stability of the formulations and lipid–DNA interaction studies were performed using transmission electron microscopy (TEM) and light scattering. TEM pictures of nanolipoplexes showed presence of two to four closely packed vesicles with signs of fusion. Efficient delivery of plasmid DNA and subsequent β-galactosidase expression was achieved using the anionic nanolipoplexes. Transfection efficiency increased with lipid:DNA ratio up to 7:1 (w/w), where transfection efficiency was 12-fold higher than in untreated cells. Further increase in lipid ratio decreased transfection. These nanolipoplexes appear to be safe, stable and efficient in the protection and delivery of DNA to different cells and tissues.     

Spectroscopic and Computational Study of a Nonheme Iron Nitrosyl Center in a Biosynthetic Model of Nitric Oxide Reductase

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme Fe_B center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the Fe_B nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}⁷ complex in a protein‐based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}⁷ complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S= 1/2) [Fe²⁺‐NO^.]. The radical nature of the Fe_B‐bound NO would facilitate N? N bond formation by radical coupling with the heme‐bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.     

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of ?32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.     

Gradient-index fiber-optic microprobes for minimally invasive in vivo low-coherence interferometry

We describe the design, construction, and application of what are believed to be the smallest fiber-optic probes used to date during imaging or diagnosis involving low-coherence interferometry (LCI). The probes use novel fiber-optic gradient-index (GRIN) lenses fabricated by a recently developed modified chemical-vapor-deposition (MCVD) process that avoids on-axis aberrations commonly marring MCVD-fabricated GRIN substrate. Fusing GRIN fiber lenses onto single-mode fiber yields automatically aligned all-fiber probes that insert into tissue through hypodermic needles as small as 31-gauge (inner diameter, 127 mum). We demonstrate the use of such probes with LCI by measuring microscopic brain motions in vivo.     

A Bound on the Total Chromatic Number

. The proof is probabilistic. Received: November 26, 1996     

The use of surface photovoltage measurements for the surface characterisation of photoconductors

Measurements of the surface photovoltage as a function of wavelength of the exciting radiation show a number of features which change with variations in the surface conditions. In the case of photoconducting cadmium sulphide, it is shown that the changing spectral features, rather than being directly associated with surface states, are due to bulk transitions which give rise to changes in the bulk carrier concentrations and (indirectly) to changes in the surface potential as a result of redistribution of charge between bulk and surface states.     

Temporal onset-order discrimination through the tactual sense

Tactual temporal-onset order thresholds were measured for two sinusoidal vibrations of different frequencies delivered to two separate locations (thumb and index finger) of a multi-finger tactual stimulating device. The frequency delivered to the thumb was fixed at 50 Hz and that to the index finger at 250 Hz. The amplitude and duration of each of the two sinusoidal vibrations were roved independently from trial to trial in a 1-interval, 2AFC procedure. Performance, measured as a function of stimulus-onset asynchrony (SOA), indicated that the temporal-onset-order threshold averaged 34 ms across four subjects. The data were further classified into subsets according to both the amplitude and duration of the two stimuli in each trial of the roving-discrimination paradigm. The results indicated that the amplitude differences of the two stimuli in each trial had a substantial effect on onset-order discrimination, while duration differences generally had little effect. The effects of amplitude differences are explained qualitatively in terms of amplitude latency relationships and stimulus interactions such as temporal masking. Overall, the results not only contribute to an enhanced understanding of the temporal sensitivity of the tactual system but also provide guidelines for the design of tactual aids for hearing-impaired persons.     

Anionic O --> alpha- and beta-vinyl carbamoyl translocation of 2-(O-carbamoyl) stilbenes

[reaction: see text] New anionic oxygen to alpha- and beta-vinyl carbamoyl migration reactions, 17a and 26a-c --> 18 and 30a-c, proceed under LDA-mediated conditions leading stereoselectively to highly substituted stilbenes bearing electron-donating and -withdrawing substituents. Compounds 17a and 26a-c are prepared by combination of efficient, directed ortho metalation, Sonogashira, and Suzuki-Miyaura cross-coupling procedures.