Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.     

Water uptake in the cat flea Ctenocephalides felis (Pulicidae: Siphonaptera)

To counteract water loss due to excretion, cuticular transpiration and respiration, various groups of arthropods have developed mechanisms for active uptake of water vapor from unsaturated air. In this study, active uptake capabilities and water loss rates were examined in the various developmental stages of the cat flea, Ctenocephalides felis. To determine critical equilibrium humidity, the lowest relative humidity at which active water uptake can occur, pre-desiccated immature and adult fleas were placed in a series of humidity regimes ranging from 44 to 93% RH. Active uptake occurred in larval stages at relative humidities above 53% and in pre-pupae at 75-93% RH. Pupae and adults did not demonstrate active uptake at any humidity. Optimal uptake for larvae occurred between 20 and 30 degrees C. When placed over Drierite (<10% RH), larval and adult stages demonstrated a higher rate of water loss than pre-pupal and pupal stages. Active water uptake is necessary to ensure proper development of the larvae of C. felis. Active uptake ceases after the larval-pupal ecdysis and it appears that adults have lost the ability to actively uptake water.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Bacteria detection using disposable optical leaky waveguide sensors

Novel disposable absorbing material clad leaky waveguide sensor devices (LWD) have been developed for the detection of pathogenic particles such as bacteria. These chips are tailored to give the maximum extension of the evanescent field at the sensor surface in order to place the entire volume of the bacteria captured by immobilized antibodies on the chip surface within this field. This in turn increases the interaction of the light with the bacteria's bulk volume. Disposable LWD chips were fabricated at room temperature and without the use of expensive fabrication equipment. These LWDs have been characterised by detecting refractive index (RI) changes, scattering and fluorescence from bacterial spores at the sensor surface when illuminated at the coupling angle. The detection limit of Bacillus subtilis var. niger (BG) bacterial spores was 10(4) spores/ml and the illumination intensity of the spores was found to be three times greater than the illumination intensity generated using the surface plasmon resonance (SPR).     

Application of beta-irradiation through the struts of a previously deployed stent

The application of beta-radiation in coronary arteries is a promising new technique for the treatment of in-stent restenosis. This is the first case in which the 5 F. delivery catheter of the Beta-Cath trade mark system was advanced through the struts of a stent, previously deployed in an adjacent branch, so as to deliver radiation to the target vessel.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Morphological differences among egg nests and adult individuals of Cicadatra persica (Hemiptera,Cicadidae), distributed in Erneh,Syria

The aim of this study is determining the different patterns of egg nests and the morphological differences between the specimens of Cicadatra persica Kirkalidy, 1909 (Hemiptera: Cicadidae) distributed in fruit orchards in Erneh located on AL-Sheikh mountain south west of Syria. The appearance of 80 egg nests was studied, and the results showed that there were two basic patterns of egg nests laid by Cicadatra persica, 90% of the egg nests were of the first pattern (consists of several adjacent slits), while 10% of them were of the second pattern (consists of several divergent slits). A random sample consisting of 300 specimens (150 males and 150 females) were also studied concentrating on the differences in the color of the supra-antennal plate and in the number of spurs on the tibia of the hind legs. The results showed that there were two basic patterns of individuals based on the differences in the color of supra-antennal plate. The first pattern (individuals with yellow supra-antennal plates), constituted more than 90%, and the second one (individuals with black supra-antennal plates) constituted less than 10%. The results also showed that there were 27 different patterns based on the number of spurs on the tibia of the hind legs. One of them was a common pattern (2, 3) whose individuals have 2 spurs on the upper side of the tibia of the hind legs and 3 spurs on the lateral side of the tibia of the hind legs. The total percent of this common pattern was 76%. The other 26 patterns were different from each other, and the total percent of all these different patterns was 24%.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.