Realized evolvability: quantifying phenotypic evolution in a Drosophila clade

Understanding the evolutionary potential of morphological evolution is still a major problem in evolutionary biology. In this study, we tried to quantify the amount of variation of different traits among species of a Drosophila clade reared under standard conditions. Nineteen different traits have been measured on nine species of the same clade, the Neotropical saltans group of Drosophila. Measured traits can be distributed into five categories: size‐traits (wing and thorax), shape indices (ratios among the size traits), number of sternopleural bristles on the thorax, number of abdominal bristles on successive sternites, and dorsal pigmentation of abdomen. All species are of medium size with a generally dark pigmentation. A remarkable feature is the presence of numerous bristles on the 6th sternite of the males, while this segment is bare in other Drosophila species. A multivariate analysis revealed that it was possible to discriminate all the investigated species by using the complete set of measured traits. For each trait, phenotypic variability was investigated at the within‐ and between‐species levels. As a rule, the interspecific coefficient of variation (CV) was much greater than the within species CV, and it is proposed to call it realized evolvability. All possible correlations were calculated between traits across species, providing many unexpected results. Size traits were highly correlated among them, but not correlated with shape indices. Abdominal traits (bristles and pigmentation) were correlated, but often in opposite directions, with thoracic shape indices. Tergite pigmentation was negatively correlated with bristle number on sternite. For the moment, most of the correlations cannot be explained by developmental processes or parallel selective pressures. Nonetheless, mapping the evolution of the two characters on a molecular phylogeny of the studied species revealed two opposite phylogenetic trends for abdominal pigmentation and setation, respectively. Our data suggest a need for similar studies in other well‐defined Drosophila clades.     

Phylogenetic placement and population structure of Indo‐Pacific bottlenose dolphins (Tursiops aduncus) off Zanzibar,Tanzania, based on mtDNA sequences

Phylogenetic placement of bottlenose dolphins from Zanzibar, East Africa and putative population differentiation between animals found off southern and northern Zanzibar were examined using variation in mtDNA control region sequences. Samples (n= 45) from animals bycaught in fishing gear and skin biopsies collected during boat surveys were compared to published sequences (n= 173) of Indo‐Pacific bottlenose dolphin, Tursiops aduncus, from southeast Australian waters, Chinese/Indonesian waters, and South African waters (which recently was proposed as a new species) and to published sequences of common bottlenose dolphin, Tursiops truncatus. Bayesian and maximum parsimony analyses indicated a close relationship between Zanzibar and South African haplotypes, which are differentiated from both Chinese/Indonesian and Australian T. aduncus haplotypes. Our results suggest that the dolphins found off Zanzibar should be classified as T. aduncus alongside the South African animals. Further, analyses of genetic differentiation showed significant separation between the T. aduncus found off northern and southern Zanzibar despite the relatively short distance (approximately 80 km) between these areas. Much less differentiation was found between southern Zanzibar and South Africa, suggesting a more recent common evolutionary history for these populations than for the northern and southern Zanzibar populations.     

3-substitued indoles: one-pot synthesis and evaluation of anticancer and Src kinase inhibitory activities

An efficient and economical method was developed for the synthesis of 3-substituted indoles by one-pot three-component coupling reaction of a substituted or unsubstituted benzaldehyde, N-methylaniline, and indole or N-methylindole using Yb(OTf)₃-SiO₂ as a catalyst. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adenocarcinoma (SK-OV-3), and c-Src kinase activity. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl (4q) indole derivatives inhibited the cell proliferation of SK-OV-3 and HT-29 cells by 70-77% at a concentration of 50 μM. The unsubstituted phenyl (4d) and 3-nitrophenyl (4l) derivatives showed the inhibition of c-Src kinase with IC₅₀ values of 50.6 and 58.3 μM, respectively.     

Development of Controlled-Release Matrix Tablet of Risperidone: Influence of Methocel®- and Ethocel®-Based Novel Polymeric Blend on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Bioavailability

Controlled-release (CR) matrix tablet of 4 mg risperidone was developed using flow bound dry granulation–slugging method to improve its safety profile and compliance. Model formulations F1, F2, and F3, consisting of distinct blends of Methocel® K100 LV-CR and Ethocel® standard 7FP premium, were slugged. Each batch of granules (250–1,000 μm), obtained by crushing the slugs, was divided into three portions after lubrication and then compressed to 9-, 12-, and 15-kg hard tablets. In vitro drug release studies were carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) using a paddle dissolution apparatus run at 50 rpm. The CR test tablet, containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness, exhibited pH-independent zero-order release kinetics for 24 h. The drug release rate was inversely proportional to the content of Ethocel®, while the gel layer formed of Methocel® helped in maintaining the integrity of the matrix. Changes in the hardness of tablet did not affect the release kinetics. The tablets were reproducible and stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. Risperidone and its active metabolite, 9-hydroxyrisperidone, present in the pooled rabbit’s serum, were analyzed with HPLC-UV at λ_max 280 nm. The CR test tablet exhibited bioequivalence to reference conventional tablet in addition to the significantly (p < 0.05) optimized peak concentration, C_max, and extended peak time, T_max, of the active moiety. There was a good association between drug absorption in vivo and drug release in vitro (R² = 0.7293). The successfully developed CR test tablet may be used for better therapeutic outcomes of risperidone.KEY WORDS: controlled release, dry granulation slugging method, risperidone     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Evaluation of epithelial mesenchymal transition in patients with chronic obstructive pulmonary disease

Background

The reticular basement membrane (Rbm) in smokers and especially smokers with COPD is fragmented with "clefts" containing cells staining for the collagenase matrix-metalloproteinase-9 (MMP-9) and fibroblast protein, S100A4. These cells are also present in the basal epithelium. Such changes are likely hallmarks of epithelial mesenchymal transition (EMT). We aimed to confirm the epithelial origin of these Rbm cells, and to exclude potential confounding by infiltrating inflammatory cells.

Methods

Endobronchial biopsy sections from 17 COPD current smokers, with documented Rbm splitting and cellularity were stained for neutrophil elastase (neutrophil marker), CD68 (macrophage/mature fibroblasts), CD4+/CD8+ T lymphocytes, CD19 (B-cells), CD11c (dendritic cells/inflammatory cells), and S100 (Langerhans cells). The number of cells in the Rbm and epithelium staining for these "inflammatory" cell markers were then compared to numbers staining for S100A4, "a documented EMT epitope". Slides were double stained for S100A4 and cytokeratin(s).

Results

In the basal epithelium significantly more cells stained for S100A4 compared to infiltrating macrophages, fibroblasts or immune cells: median, 26 (21.3 - 37.3) versus 0 (0 - 9.6) per mm, p < 0.003. Markedly more S100A4 staining cells were also observed in the Rbm compared to infiltrating macrophages, neutrophils, fibroblasts or immune cells or any sub-type: 58 (37.3 - 92.6) versus 0 (0 - 4.8) cells/mm Rbm, p < 0.003. Cells in the basal epithelium 26 (21.3 - 37.3) per mm) and Rbm (5.9 (2.3 - 13.8) per mm) frequently double stained for both cytokeratin and S100A4.

Conclusions

These data provide additional support for active EMT in COPD airways.     

An effective extracellular protein secretion by an ABC transporter system in <Emphasis Type="Italic">Escherichia coli</Emphasis>: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production

Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.