Development and Validation of a Discriminative Dissolution Test for Betamethasone Sodium Phosphate and Betamethasone Dipropionate Intramuscular Injectable Suspension

The intramuscular administration of the injectable suspension betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BD) has immediate therapeutic activity due to solubilized BSP and prolonged activity resulting from the slow release of BD micro-crystals. The purpose of this study was to develop and validate a dissolution method for BD in intramuscular injectable suspensions with detection by high-performance liquid chromatography (HPLC) method. Five commercial products presented a distribution of particle sizes, ranging between 7.43 and 40.25 μm as measured by laser diffraction. It was also found that particle sizes differed between batches of the same product. The different products were tested using the paddle apparatus, with stirring speeds of 25 and 50 rpm in 300 mL of phosphate buffer; simulated body fluid, muscle fluid, and synovial fluid were used as biorelevant dissolution media at 37 ± 0.5°C. It was verified that not only does average particle size affect the dissolution rate, but also the mode and the polydispersity index of the particles. Discriminatory power was obtained using the in vitro dissolution method with 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% sodium lauryl sulfate and a stirring speed of 50 rpm. The HPLC-method is linear, precise, selective, and accurate for the quantification of BSP and BD in dissolution profile testing. This dissolution method can be utilized as a method to control the quality of these injectable suspensions.Key words: dipropionate betamethasone, dissolution test, intramuscular injectable suspensions, simulated muscular fluid, sodium phosphate betamethasone     

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

The core of affects: namer and named in Bahia (Brazil)

This article is a study of personal naming using drawings made by secondary school students from two small towns in coastal Bahia (Brazil) of the persons who gave them their names. The article explores the relation between namer and named as one of continued identity created by imaginative processes of surrogation akin to pretend‐play. In early ontogeny, the person is constituted through a series of mutualities of being that come to constitute a core of affects. The article aims to contribute towards a better understanding of the processes of constitution of personhood in the context of human relatedness.     

Protective effect of mannan oligosaccharides against early colonization by Salmonella Enteritidis in chicks is improved by higher dietary threonine levels

Aims

To evaluate mannan oligosaccharide (MOS) and threonine effects on performance, small intestine morphology and Salmonella spp. counts in Salmonella Enteritidis‐challenged birds.

Methods and Results

One‐day‐old chicks (1d) were distributed into five treatments: nonchallenged animals fed basal diet (RB‐0), animals fed basal diet and infected with Salmonella Enteritidis (RB‐I), animals fed high level of threonine and infected (HT‐I), birds fed basal diet with MOS and infected (MOS‐I), birds fed high level of threonine and MOS and infected (HT+MOS‐I). Birds were inoculated at 2d with Salmonella Enteritidis, except RB‐0 birds. Chicks fed higher dietary threonine and MOS showed performance similar to RB‐0 and intestinal morphology recovery at 8 dpi. Salmonella counts and the number of Salmonella‐positive animals were lower in HT+MOS‐I compared with other challenged groups.

Conclusion

Mannan oligosaccharides and threonine act synergistically, resulting in improved intestinal environment and recovery after Salmonella inoculation.

Significance and Impact of the Study

Nutritional approaches may be useful to prevent Salmonella infection in the first week and putative carcass contamination at slaughter. This is the first report on the possible synergistic effect of mannan oligosaccharides and threonine, and further studies should be performed including performance, microbiota evaluation, composition of intestinal mucins and immune assessment.     

Characterization,subsite mapping and N-terminal sequence of miliin,a serine-protease isolated from the latex of Euphorbia milii

Miliin is a serine protease purified from the latex of Euphorbia milii. This work reports the effect of pH and temperature on the catalytic activity of miliin, using fluorescence resonance energy transfer (FRET) substrates. Miliin displayed the highest activity at pH 9 and 35 °C. Subsite mapping shows that subsites S₂ to S₂′ prefer uncharged residues. The S₂ subsite prefers hydrophobic aliphatic amino acids (Val, Pro and Ile) and defines the cleavage site. This work is the first one that reports subsite mapping of Euphorbiacea proteases. The N-terminal sequence showed higher similarity (40%) with the serine protease LIM9 isolated from Lilium. The presence of Tyr, Pro and Lys at positions 2, 5 and 10 respectively, were observed for most of the serine proteases used for comparison. The N-terminal sequence has striking differences with those reported previously for milin and eumiliin, other serine proteases isolated from the latex of E. milii.     

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

Aims/hypothesis

Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR^−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca^2 + handling in these islets.

Methods

Isolated islets from both LDLR^−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca^2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).

Results

The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR^−/− than in WT islets, paralleled by an impairment of Ca^2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR^−/− compared with WT islets. Removal of excess cholesterol from LDLR^−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca^2 + handling was also normalized in cholesterol-depleted LDLR^−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca^2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.

Conclusion

Abnormally high (LDLR^−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca^2 + handling. Normalization of cholesterol improves Ca^2 + handling and insulin secretion in LDLR^−/− islets.     

Reproductive strategy of the invasive Oxalis pes-caprae: distribution patterns of floral morphs, ploidy levels and sexual reproduction

Oxalis pes-caprae, a tristylous flowering plant native to South Africa, is described in the western Mediterranean basin as an asexual—only 5x short-styled morph (5x S-morph) invasive weed losing all mating partners after introduction. The objective of this study was to reassess the patterns of floral morph and cytotype distribution and the sexual reproduction ability in this invaded range. For that, floral morph and cytotype composition were evaluated in 39 populations of O. pes-caprae in a methodical sampling. The reproductive success of natural populations was assessed as fruit and seed production and seed germination for all floral morphs and cytotypes detected. Self- and morph-incompatibility were also studied with controlled hand pollinations. A remarkable diversity in floral morph and cytotype composition was observed. Furthermore, we observed successful sexual reproduction in several localities across the surveyed area. The S-morph is still dominant in this invaded area, and although it was mostly 5x, an additional cytotype (4x) was also recorded. Records of both a mid-styled morph (M-morph) and an area with trimorphic populations of this species are reported here for the first time in the invasive range of the Mediterranean basin. The long-styled morph appears to occur randomly across the surveyed area, while the M-morph is concentrated mainly in Estremadura province (Portugal), where a breakdown in the incompatibility system was observed. These distribution patterns may result from events of sexual reproduction after incompatibility breakdown and/or from multiple introduction events from the native area. The ability to reproduce sexually, undetected so far, may have important impacts in the population dynamics and major consequences for the adaptation and selection potential of O. pes-caprae in this invaded area.     

Assessment of the total effective xenoestrogen burden in extracts of human placentas

We have standardized a method to assess the total effective xenoestrogen burden (TEXB) in human placentas by the extraction and separation by high-performance liquid chromatography of two fractions containing lipophilic xenoestrogens (alpha) and endogenous hormones (beta), followed by assessing their estrogenicity in MCF-7 breast cancer cell-based E-Screen and Yeast Estrogen Screen (YES) bioassays. The means of TEXB alpha concentrations (in estradiol equivalent (Eeq) units) were 1.32 and 0.77 Eeq pM g^?1 placenta in the E-Screen and YES, respectively; TEXB beta concentrations were 6.97 and 11.56 Eeq pM g^?1 placenta, respectively. The interclass correlation coefficient was low and a fair level of agreement was observed after kappa test correction. According to the E-Screen and YES, TEXB alpha was ≥LOD in 70.0 and 55.0% of the placentas and 92.5 and 82.5% in beta, respectively. Although both bioassays can be recommended for assessing TEXB, there is greater experience with the use of the E-Screen for estrogenic assessment after extensive extraction of complex human matrices.     

Differentially expressed proteins during an incompatible interaction between common bean and the fungus Pseudocercospora griseola

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.