Comparison of Three Dissolution Apparatuses for Testing Calcium Phosphate Pellets used as Ibuprofen Delivery Systems

Porous calcium phosphate pellets were produced according to two granulation processes (low and high shear wet granulations) and drug loaded with five ibuprofen contents (1.75%, 7%, 12.5%, 22%, and 36%) in order to ensure both bone defect filling and local drug delivery. The drug-release kinetics from the two types of pellets was studied using three dissolution apparatuses: paddle apparatus, reciprocating cylinder, and flow-through cell. The paper compared the three dissolution methods and considered the effect of the granulation process on the ibuprofen-release kinetics. Dissolution data were analyzed using the Weibull function as well as the difference (f1) and similarity (f2) factors. Dissolution kinetics was not influenced by the granulation process, regardless of the dissolution apparatus and of the drug content. The comparison of the three dissolution devices indicated that ibuprofen was released faster from granules loaded with 36% of drug content with the reciprocating apparatus, due to the disintegration of the granules occurring during the dissolution test. For the other drug contents, dissolution profiles were not significantly different from one apparatus to another. However, the flow-through cell seemed to be more suitable for the drug-release study of implantable materials.     

Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.     

Foraging behavior of Xylocopa (Neoxylocopa) cearensis Ducke (Hymenoptera: Apidae, Xylocopini) in a population of Cuphea brachiata Koehne (Lythraceae)

An analysis of the foraging behavior of the bee Xylocopa (Neoxylocopa) cearensis Ducke among shrubs of Cuphea brachiata Koehne (Lythraceae), a key component in the Abaeté coastal sand dunes, Salvador, BA, Brazil, suggests that this bee is very important for the maintenance of the plant population, performing the pollination. This dispersal, however, is spatially restricted, so the populations in the area are likely to be highly structured genetically.     

Methylomic analysis of monozygotic twins discordant for childhood psychotic symptoms

Childhood psychotic symptoms are associated with increased rates of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood; thus, elucidating early risk indicators is crucial to target prevention efforts. There is considerable discordance for psychotic symptoms between monozygotic twins, indicating that child-specific non-genetic factors must be involved. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to childhood psychotic symptoms. Therefore, this study explored whether differences in DNA methylation at age 10 were associated with monozygotic twin discordance for psychotic symptoms at age 12. The Environmental Risk (E-Risk) Longitudinal Twin Study cohort of 2,232 children (1,116 twin pairs) was assessed for age-12 psychotic symptoms and 24 monozygotic twin pairs discordant for symptoms were identified for methylomic comparison. Children provided buccal samples at ages 5 and 10. DNA was bisulfite modified and DNA methylation was quantified using the Infinium HumanMethylation450 array. Differentially methylated positions (DMPs) associated with psychotic symptoms were subsequently tested in post-mortem prefrontal cortex tissue from adult schizophrenia patients and age-matched controls. Site-specific DNA methylation differences were observed at age 10 between monozygotic twins discordant for age-12 psychotic symptoms. Similar DMPs were not found at age 5. The top-ranked psychosis-associated DMP (cg23933044), located in the promoter of the C5ORF42 gene, was also hypomethylated in post-mortem prefrontal cortex brain tissue from schizophrenia patients compared to unaffected controls. These data tentatively suggest that epigenetic variation in peripheral tissue is associated with childhood psychotic symptoms and may indicate susceptibility to schizophrenia and other mental health problems.     

Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality.     

Morphometric analysis and taxonomic revision of the Vriesea paraibica complex (Bromeliaceae)

The species related to Vriesea paraibica (Bromeliaceae, Tillandsioideae) have controversial taxonomic limits. For several decades, this group has been identified in herbarium collections as V. × morreniana, an artificial hybrid that does not grow in natural habitats. The aim of this study was to assess the morphological variation in the V. paraibica complex through morphometric analyses of natural populations. Two sets of analyses were performed: the first involved six natural populations (G1) and the second was carried out on taxa that emerged from the first analysis, but using material from herbarium collections (G2). Univariate ANOVA was used, as well as discriminant analysis of 16 morphometric variables in G1 and 18 in G2. The results of the analyses of the two groups were similar and led to the selection of diagnostic traits of four species. Lengths of the lower and median floral bracts were significant for the separation of red and yellow floral bracts. Vriesea paraibica and V. interrogatoria have red bracts; these two species are differentiated by the widths of the lower and median portions of the inflorescence and by scape length. These structures are larger in the former and smaller in the latter. Of the species with yellow floral bracts, V. eltoniana is distinguished by longer leaf blades and scapes and V. flava is characterized by its shorter sepal lengths. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 163–181.     

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus × Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n = 12) or during a shortened cycle, with luteolysis induction 10 d after estrus (Experiment 2; n = 6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10 d after estrus (P < 0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r = 0.86; P < 0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24 h compared with 72 h; P < 0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P > 0.70), but a day effect on this attribute was observed in Experiment 2 (P = 0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r = −0.63; P < 0.05), but not in Experiment 2 (r = −0.28; P > 0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P < 0.009) and luteolysis-induced (Experiment 2; P < 0.05) estrous cycles (P < 0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r = −0.71 and −0.58 in Experiments 1 and 2, respectively; P < 0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.     

Pregnancy rates and corpus luteum-related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus × Bos taurus indicus) nonlactating cows and heifers (n = 259) were treated with the following protocol: 2 mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9 g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F_2α (PGF_2α) and CIDR withdrawal (Day 8); and 1 mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n = 90) or in vitro-produced (n = 87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P < 0.001). Mean (±SD) plasma progesterone (P₄) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2 ± 5.0 vs. 3.8 ± 2.4 ng/mL; P = 0.02). Mean pixel values (71.8 ± 1.3 vs. 71.2 ± 1.1) and pixel heterogeneity (14.8 ± 0.3 vs. 14.5 ± 0.5) were similar between pregnant and nonpregnant recipients (P > 0.10). No significant relationship was detected between pregnancy outcome and plasma P₄, corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.     

Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.Bacillus sphaericus is considered an important entomopathogen due to its capacity to produce insecticidal proteins with specific action against mosquitoes (Diptera: Culicidae). The binary (Bin) toxin, which is produced during bacterial sporulation and deposited in parasporal crystalline inclusions, is the most important larvicidal factor. Other proteins characterized, such as mosquitocidal toxins (Mtx proteins), can be produced during vegetative growth, and although these proteins may have larvicidal potential, they play a minor role in the toxicity of the native strains since they are produced by vegetative cells and are degraded by B. sphaericus proteinases (20, 30), and do not form components of the spore-crystal preparations that are used in control programs. Recently, a new two-component toxin was characterized from B. sphaericus strain IAB59. This is formed by the proteins Cry48Aa (135 kDa) and Cry49Aa (53 kDa), which are produced as crystalline inclusions (13). The toxin has a unique composition since the Cry48Aa component belongs to the three-domain family of Cry proteins with 30% similarity to the mosquitocidal Cry4Aa protein from Bacillus thuringiensis serovar israelensis, while Cry49Aa is one of the Bin-toxin-like proteins, a family that comprises the Bin toxin from B. sphaericus, in addition to the Cry36 and Cry35 proteins from B. thuringiensis (9, 13).Cry48Aa/Cry49Aa is considered a two-component toxin because neither component shows toxicity alone, whereas both can act in synergy and the optimum level of toxicity to Culex species is achieved when the two are present at an equimolar ratio. The 50% lethal concentration for third-instar larvae equates to 15.9 ng/ml Cry48Aa and 6.3 ng/ml Cry49Aa of purified toxins, which is a level of toxicity comparable to that of the Bin toxin (13). However, in contrast to the Bin toxin, which is naturally produced in an equimolar ratio, Cry48Aa production is low in native strains and does not confer high toxicity (13). The initial steps of the mode of action of Bin and Cry48Aa/Cry49Aa crystals are similar and comprise the ingestion of crystals, solubilization under alkaline pH, and activation of protoxins into toxins by midgut proteases. After processing, Bin toxin recognizes and binds to specific receptors in the midgut of Bin-toxin-susceptible species through its subunit BinB (51 kDa), while the component BinA (42 kDa) confers toxicity and is likely to form pores in the cell membrane (7, 25). The membrane-bound receptors of Bin toxin on the midgut of Culex quinquefasciatus larvae, Cqm1, were characterized as 60-kDa α-glucosidases (24). The mode of action of Cry48Aa/Cry49Aa is still unknown, but a remarkable feature of this new two-component toxin is the capacity to overcome C. quinquefasciatus resistance to the Bin toxin (13, 19, 21). Resistance of Culex larvae to the Bin-toxin-based larvicides often relies on the absence of functional Cqm1 receptors in the midgut (19, 24, 26). As a consequence, toxins with a distinct mode of action, such as Cry48Aa/Cry49Aa as well as B. thuringiensis serovar israelensis toxins (Cry11Aa, Cry4Aa, Cry4Ba, and Cyt1Aa), do not experience cross-resistance in the Bin-toxin-resistant larvae (12, 21, 32). Such toxins can play a strategic role in the management of resistance, and the major goal of this study was to investigate the ultrastructural effects of the Cry48Aa/Cry49Aa toxin on Bin-toxin-susceptible and -resistant C. quinquefasciatus larvae and to compare these with the effects of a synergistic mixture of Bin/Cry11Aa used to overcome Bin toxin resistance.