Comparative Evaluation of Porous Versus Nonporous Mucoadhesive Films as Buccal Delivery System of Glibenclamide

The present research work focused on the comparative assessment of porous versus nonporous films in order to develop a suitable buccoadhesive device for the delivery of glibenclamide. Both films were prepared by solvent casting technique using the 3² full factorial design, developing nine formulations (F1–F9). The films were evaluated for ex vivo mucoadhesive force, ex vivo mucoadhesion time, in vitro drug release (using a modified flow-through drug release apparatus), and ex vivo drug permeation. The mucoadhesive force, mucoadhesion time, swelling index, and tensile strength were observed to be directly proportional to the content of HPMC K4M. The optimized porous film (F4) showed an in vitro drug release of 84.47 ± 0.98%, ex vivo mucoadhesive force of 0.24 ± 0.04 N, and ex vivo mucoadhesion time of 539.11 ± 3.05 min, while the nonporous film (NF4) with the same polymer composition showed a release of 62.66 ± 0.87%, mucoadhesive force of 0.20 ± 0.05 N, and mucoadhesive time of 510 ± 2.00 min. The porous film showed significant differences for drug release and mucoadhesion time (p < 0.05) versus the nonporous film. The mechanism of drug release was observed to follow non-Fickian diffusion (0.1 < n < 0.5) for both porous and nonporous films. Ex vivo permeation studies through chicken buccal mucosa indicated improved drug permeation in porous films versus nonporous films. The present investigation established porous films to be a cost-effective buccoadhesive delivery system of glibenclamide.KEY WORDS: buccoadhesive drug delivery, glibenclamide, in vitro release and ex vivo permeation, porous film     

Comparison of structure,regeneration and dead wood in virgin forest remnant and managed forest on Grmeč Mountain in Western Bosnia

This paper compares the forest structure, regeneration and distribution of dead wood in a virgin forest remnant and a close-to-nature managed beech–conifer mixture situated on Grme? Mountain in Western Bosnia. The investigations were carried out in a 1 ha permanent sample plot and 35 circular plots (20 m radius) in the virgin forest and in 17 circular plots (25 m radius) in managed forests. The number of trees in the managed forest was significantly (p = 0.05) higher than that in virgin forest and the distribution of the number of trees per diameter classes had a decreasing trend, but with a different shape in the virgin forest compared to the managed stands. In the lower diameter classes, the stock volume recorded in virgin forest was half of that in the managed forest, whilst for higher diameter classes the cumulated volume of the growing stock was almost double in virgin forest. The young crops had a significantly lower presence in the virgin forest and a larger volume of dead wood was identified in the virgin forest than in managed stands. The study results are important in assessing the consequences of close-to-nature management on the forest structure and regeneration when compared to the condition in virgin forests.     

Development of a set of genomic microsatellite markers in tea (Camellia L.) (Camelliaceae)

Tea (Camellia L.) is the world’s most consumed health drink and is also important economically. Due to its self-incompatible and outcrossing nature, tea is composed of highly heterogeneous germplasm. It is a perennial, slow-growing crop and hence the successful release of new improved cultivars following conventional breeding methods takes years. In this context, a DNA marker-based molecular breeding approach holds great promise in accelerating genetic improvement programs in tea. Here we describe the isolation of a set of highly polymorphic genomic microsatellite markers using the enrichment approach, which may be useful for phylogenetic and marker-assisted breeding programs in tea. The enriched library comprising 3,205 clones was screened for the presence of microsatellites using a three-primer-based colony PCR method. Four hundred positive clones were selected and sequenced, to reveal 153 sequences containing simple sequence repeats. Seventy-eight primer pairs were designed from repeat-positive sequences, out of which 40 primer pairs produced successful amplifications. Twenty-two of these primer pairs, when tested on a panel of 21 diverse tea clones and accessions, were found to be highly polymorphic, resulting in 137 alleles with an average of 6.76 alleles per primer pair. The polymorphic information content (PIC), expected heterozygosity (H _e) and observed heterozygosity (H _o) of the polymorphic markers ranged from 0.1 to 0.9, 0.1–0.9 and 0.0–0.8, with average values of 0.6 ± 0.18, 0.7 ± 0.17 and 0.5 ± 0.22, respectively. These markers can be applied for various diversity analyses, mapping programs and genotyping of tea crop.     

Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors

The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.     

Antibody-quantum dot conjugates exhibit enhanced antibacterial effect<Emphasis Type="Italic">vs.</Emphasis> unconjugated quantum dots

The effect of a 20-min exposure to antibody-quantum dot (Ab-QD) conjugates on colony counts of Escherichia coli was assessed by the spread-plate method and compared with exposure to unconjugated QDs having only amine or carboxyl groups on their surfaces. Under these conditions, Ab-QD conjugates generally exhibited >90% reduction in colony-forming units as compared to untreated E. coli and E. coli treated with unconjugated QDs after incubation for as long as 41 h. The antibacterial effect of Ab-QD conjugates vs. unconjugated QDs on Salmonella enterica subsp. enterica serovar Typhimurium was also assessed by means of a disk-diffusion technique which demonstrated greater growth inhibition (approximately 3 mm greater) by Ab-QD conjugate-impregnated disks than by unconjugated-QD-only-impregnated disks at a 10-microg disk load. At a 25-microg disk load, both treatment groups exhibited nearly equal growth inhibition.     

A modelling study of feedforward activation in human erythrocyte glycolysis

Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme's kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme's kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell's demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972-979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would otherwise occur during variations in the cell's demand for ATP. In addition, by significantly raising the steady-state setpoint of intermediate metabolite pools, the productivity index (ratio of glycolytic flux to total metabolites in the pathway) of glycolysis would fall almost four-fold in the absence of forward activation.     

Product Development Studies on Surface-Adsorbed Nanoemulsion of Olmesartan Medoxomil as a Capsular Dosage Form

The present study aimed at development of capsular dosage form of surface-adsorbed nanoemulsion (NE) of olmesartan medoxomil (OLM) so as to overcome the limitations associated with handling of liquid NEs without affecting their pharmaceutical efficacy. Selection of oil, surfactant, and cosurfactant for construction of pseudoternary phase diagrams was made on the basis of solubility of drug in these excipients. Rationally selected NE formulations were evaluated for percentage transmittance, viscosity, refractive index, globule size, zeta potential, and polydispersity index (PDI). Formulation (F3) comprising of Capmul MCM® (10% v/v), Tween 80® (11.25% v/v), polyethylene glycol 400 (3.75% v/v), and double-distilled water (75% v/v) displayed highest percentage cumulative drug release (%CDR; 96.69 ± 1.841), least globule size (17.51 ± 5.87 nm), low PDI (0.203 ± 0.032), high zeta potential (−58.93 ± 0.98 mV), and hence was selected as the optimized formulation. F3 was adsorbed over colloidal silicon dioxide (2 ml/400 mg) to produce free-flowing solid surface-adsorbed NE that presented a ready-to-fill capsule composition. Conversion of NE to surface-adsorbed NE and its reconstitution to NE did not affect the in vitro release profile of OLM as the similarity factor with respect to NE was found to be 66% and 73% respectively. The %CDR after 12 h for optimized NE, surface-adsorbed NE, and reconstituted NE was found to be 96.69 ± 0.54, 96.07 ± 1.76, and 94.78 ± 1.57, respectively (p > 0.05). The present study established capsulated surface-adsorbed NE as a viable delivery system with the potential to overcome the handling limitations of NE.KEY WORDS: bioavailability, nanoemulsion, olmesartan medoxomil, oral     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.