Fluorescence anisotropy in the frequency domain by an optical microscope

Fluorescence anisotropy decay spectroscopy is a suitable tool for investigating the size and the shape of biological molecules. We coupled this technique to an optical microscope in order to reduce the excitation volume and to allow its application to spatially inhomogeneous samples. Phase modulated measurements of the fluorescence anisotropy decay were performed by feeding an intensity modulated linearly polarized laser beam to the epifluorescence port of a microscope. Here we report the test of the dynamic response of the microscope by comparing the lifetime and fluorescence polarization anisotropy decays obtained in cuvettes in a standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor for the objective depolarization is introduced in the best-fit functions for the data analysis of the decays, the results obtained on the two setups are comparable. Some applications are reported here on long DNA tracts as well on short DNA fragments containing structural anomalies.     

Familiarity with conspecific odor and isolation-induced aggressive behavior in male mice (Mus domesticus).

60 male Swiss CD-1 mice were isolated for 24 days. In the isolation group mice were exposed to their own odor; in the familiarity group mice were familiarized with pairmates' odor by daily exchange of bedding; and in the unfamiliarity group Ss (exposed) were familiarized with pairmates' odor, whereas the pairmates (nonexposed) were familiarized with odors of other isolated mice. Aggressive behavior was scored during 20-min sessions. Familiarity with the pairmate's odor (familiarity and exposed subgroups) caused an enhancement of aggressive behavior, shown by the highest frequency of tail rattling and offensive upright posture and by a decrease in latency to the 1st attack. Nonexposed Ss showed high values of submissive upright posture, screaming, fleeing, and freezing. The results suggest that dominantlike behavior, acquired in social isolation, may be directed toward a conspecific whose odor is familiar. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Pharmaceutical excipients properties and screw feeder performance in continuous processing lines: a Quality by Design (QbD) approach

Abstract

Screw feeder performance is a critical aspect in continuous manufacturing processes. Pharmaceutical excipients, such as mannitol, microcrystalline cellulose, lactose monohydrate, and anhydrous dibasic calcium phosphate can present problems in ensuring a continuous stable feed rate due to their sub-optimal flow properties. In alignment with Quality by Design (QbD) goals, the aim of this work was to identify and explain critical sources of variability of some powder excipients delivery by screw feeding, in particular to continuous processing lines. Pharmaceutical excipients with a wide range of material properties were selected, and the impact of their flow and density properties on screw feeder performance was investigated. The analysis of the powder conveying by the screws was performed at different hopper fills and different screw speeds. A multivariable model involving bulk density (CBD) and parameters from FT4 dynamic downwards testing (SI) and dynamic upwards testing (SE) explained 95.7% of excipients feed rates (p?<?.001). The study gathers valuable information about the screw feeder performance and input materials properties that can help process understanding and QbD-based development of solid dosage forms in continuous processing lines.     

Effect of flaxseed oil in diet on fatty acid composition in the liver of Nile tilapia (Oreochromis niloticus)

This study analyzed the effects of different concentrations of flaxseed oil (FO) on the proximate composition and the contents of alpha-linolenic acid (LNA, 18:3n-3), eicosapentaenoic (EPA, 20:5n-3), and docosahexaenoic (DHA, 22:6n-3) fatty acids in the liver of cultured Nile tilapia (Oreochromis niloticus). During the five-month culture period, tilapias were given diets with incremental concentrations of FO (0.00%; 1.25%; 2.50%; 3.75%, and 5.00%) as a replacement of sunflower oil (control). There was no significant difference in moisture and ash content in the liver between treatments. Protein values ranged from 12.1% (treatment II) to 13.9% (treatment V) and total lipids ranged from 5.6% (treatment V) to 7.2% (treatment II). There was no significant difference between most treatments. Fatty acid methyl esters (FAMEs) were quantitatively analyzed by capillary gas chromatography against a C(23:0), internal standard. Variations in concentrations (in mg g(-1) of total lipids) of fatty acids between treatment I and treatment V ranged from 4.2 to 51.2 (LNA), from 0.2 to 2.3 (EPA), and from 10.6 to 56.2 (DHA), respectively. This experiment demonstrated that increasing amounts of LNA in feed may markedly increase the amounts of LNA, EPA, and DHA in the liver of Nile tilapia.     

Influence of Maternal Diet During Early Pregnancy on the Fatty Acid Profile in the Fetus at Late Pregnancy in Rats

The aim of the study was to determine the effects of different dietary fatty acids during the first half of pregnancy on the fatty acid composition of maternal adipose tissue and of maternal and fetal plasma at mid- and late-pregnancy. Pregnant rats received soybean-, olive-, fish-, linseed- or palm-oil diets from conception to day 12 of gestation. Virgin rats receiving the same treatments were studied in parallel. At day 12, some rats were sacrificed and others were returned to the standard diet and studied at day 20. At day 12, the concentrations of most fatty acids in plasma reflected the dietary composition and individual fatty acids in lumbar adipose tissue of pregnant rats correlated with those in the diet. At day 20, the plasma concentration of each fatty acid was higher in pregnant than in both virgin rats and day-12 pregnant rats. The composition in 20-day pregnant (but not in virgin) rats resembled the diet consumed during the first 12 days. Fatty acid concentration in fetal plasma was also influenced by the maternal diet during the first 12 days of pregnancy, and long-chain polyunsaturated fatty acid (LC-PUFA) concentrations correlated with those in the mothers. In conclusion, during the first half of pregnancy maternal adipose tissue stores dietary-derived fatty acids, which are released into blood during late pregnancy enabling LC-PUFA to become available to the fetus.     

Human-specific E.coli single nucleotide polymorphism (SNP) genotypes detected in a South East Queensland waterway, Australia

The World Health Organization recommends that the majority of water monitoring laboratories in the world test for E. coli daily since thermotolerant coliforms and E. coli are key indicators for risk assessment of recreational waters. Recently, we developed a new SNP method for typing E. coli strains, by which human-specific genotypes were identified. Here, we report the presence of these previously described specific SNP profiles in environmental water, sourced from the Coomera River, located in South East Queensland, Australia, over a period of two years. This study tested for the presence of human-specific E. coli to ascertain whether hydrologic and anthropogenic activity plays a key role in the pollution of the investigated watershed or whether the pollution is from other sources. We found six human-specific SNP profiles and one animal-specific SNP profile consistently across sampling sites and times. We have demonstrated that our SNP genotyping method is able to rapidly identify and characterize human- and animal-specific E. coli isolates in water sources.     

Effect of combined treatment of hydrolysis and polymerization with transglutaminase on β-lactoglobulin antigenicity

The effect of combined treatments of hydrolysis with different proteases, and subsequent polymerization with transglutaminase on the antigenic activity of β-Lg was studied. For the hydrolysis of β-Lg using Alcalase, Neutrase or bromelain, the reaction conditions were 3?% β-Lg and enzyme:substrate 25?U?g^?1 of protein, as was defined using factorial study. Under these conditions, the degree of hydrolysis (DH) of the hydrolysates was 12.6?% when obtained with Alcalase and approximately 4?% with Neutrase or bromelain. Post-hydrolysis polymerization did not result in an increase in molecular mass of the protein, but these samples presented a lower DH, determined by trinitrobenzenosulfonic acid (TNBS) method, suggesting that polymerization had occurred. Hydrolysis with the three enzymes reduced the β-Lg antigenicity, as evaluated by ELISA and immunoblotting analyses. The IgE-binding responses were practically null (<9?μg?mL^?1), 22.82 and 55.73?μg?mL^?1 towards the hydrolysates obtained with Alcalase, bromelain, and Neutrase, respectively. The post-hydrolysis polymerization increased or had no significant effect (P?≥?0.05) on the antigenic response of the hydrolysates.     

Brain Proteome and Behavioural Analysis in Wild Type,BDNF+/− and BDNF−/− Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF^+/− and knock out BDNF^−/−) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.