Sour Beer with Lacticaseibacillus paracasei subsp. paracasei F19: Feasibility and Influence of Supplementation with Spondias mombin L. Juice and/or By-Product.

This study aimed to evaluate the probiotic strain Lacticaseibacillus (L.) paracasei subsp. paracasei F19 (F19) with the yeast Saccharomyces cerevisiae US-05 (US-05), using Spondias mombin L. ('taperebá' or 'cajá') juice and by-product, in four sour-type beer formulations: control, with bagasse, juice, and juice and bagasse. The viability of F19 was evaluated by pour-plating and PMA-qPCR. Fermentability, in addition to physicochemical and sensory parameters, and aroma and flavor, were evaluated during brewery by using Headspace Solid-Phase Microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). F19 was successful in fermenting bagasse in a MRS medium (9.28 log CFU/mL in 24 h) but had a low viability in hopped wort, growing better in formulations without bagasse or juice. No difference between formulations was observed regarding sensory acceptability, and the HS-SPME/GC-MS revealed different flavors and aroma compounds. In conclusion, the production of a potential probiotic sour beer with F19 and US-05 is feasible regarding probiotic viability. However, S. mombin, as juice or bagasse, threatened probiotic survival. Different flavors and aroma compounds were detected, whereas no difference between formulations was found regarding sensory acceptability. The moderate alcohol content achieved is important for bacterial survival and for the development of a probiotic beer with health claims.

Variant expression signatures of microRNAs and protein related to growth in a crossbreed between two strains of Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed.

Time-dependent expression and activity of cytochrome P450 1s in early life-stages of the zebrafish (Danio rerio).

Zebrafish embryos are being increasingly used as model organisms for the assessment of single substances and complex environmental samples for regulatory purposes. Thus, it is essential to fully understand the xenobiotic metabolism during the different life-stages of early development. The aim of the present study was to determine arylhydrocarbon receptor (AhR)-mediated activity during selected times of early development using qPCR, enzymatic activity through measurement of 7-ethoxyresorufin-O-deethylase (EROD) activity, and protein expression analysis. In the present study, gene expression of cyp1a, cyp1b1, cyp1c1, cyp1c2, and ahr2 as well as EROD activity were investigated up to 120 h postfertilization (hpf) after exposure to either ß-naphthoflavone (BNF) or a polycyclic aromatic hydrocarbons (PAH)-contaminated sediment extract from Vering Kanal in Hamburg (VK). Protein expression was measured at 72 hpf after exposure to 20 µg/L BNF. Altered proteins were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting. Distinct patterns of basal messenger RNA (mRNA) expression were found for each of the cyp1 genes, suggesting specific roles during embryonic development. All transcripts were induced by BNF and VK. ahr2 mRNA expression was significantly upregulated after exposure to VK. All cyp1 genes investigated showed a temporal decline in expression at 72 hpf. The significant decline of Hsp 90ß protein at 72 hpf after exposure to BNF may suggest an explanation for the decline of cyp1 genes at this time point as Hsp 90ß is of major importance for the functioning of the Ah-receptor. EROD activity measured in embryos was significantly induced after 96 hpf of exposure to BNF or VK. Together, these results demonstrate distinct temporal patterns of cyp1 genes and protein activities in zebrafish embryos as well as show a need to investigate further the xenobiotic biotransformation system during early development of zebrafish.

Effects of thawing, refreezing and storage conditions of tissue samples and protein extracts on 2-DE spot intensity.

We report that reliable quantitative proteome analyses can be performed with tissue samples stored at -80 degrees C for up to 10 years. However, storing protein extracts at 4 degrees C for 24 h and freezing protein extracts at -80 degrees C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2-DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2-DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.

In-Gel 18O labeling for improved identification of proteins from 2-DE Gel spots in comparative proteomic experiments.

The reliability of 2-DE gel-based comparative proteomics is severely impaired by the potential presence of overlapping proteins. We describe a methodological procedure which may solve this problem. Corresponding protein spots from two experimental groups are digested in the presence of 16O and 18O, respectively. Samples are pooled and proteins identified by MS. The 18O/16O-ratios of the different proteins found in the same spot distinguish proteins with altered from those whose intensity is unchanged.

Strategies for a reliable biostatistical analysis of differentially expressed spots from two-dimensional electrophoresis gels.

We performed quantitative comparisons with the two-dimensional gel electrophoresis technique and evaluated the reliability of biostatistical tests for the correction of "false significant" results (alpha-error) by performing repeated runs of an experiment. Results based on uncorrected p-values yielded numerous significant differences in spot intensity which could not be replicated in two additional runs. The best strategy for avoiding these "false-positive" results was strongly dependent on the type of result. In experiments yielding very marked group differences in spot intensity, calculation of the "False Discovery Rate" (FDR) by the Benjamini and Hochberg method corrected the results with sufficient reliability. In experiments yielding relatively small (p-values>0.001) group differences, up to 100% of all results which were significant in two repeated runs were excluded ("false-negative") by calculation of the FDR. In such experiments, significant differences need confirmation by repeated runs.

Results and reliability of protein quantification for two-dimensional gel electrophoresis strongly depend on the type of protein sample and the method employed.

We investigated the effects of tissue samples taken from rat brain on the reliability of three protein quantification kits: the Bradford assay, the 2-D Quant Kit, and the EZQ Protein Quantitation Kit. All three assays measured significantly smaller amounts of protein after extraction than the reference values before extraction. Only small effects were seen in homogenates, but very pronounced differences in membrane-enriched and highly lipophilic subcellular fractions. Researchers should evaluate which method of protein quantification is best qualified for their specific experimental design.

The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis.

Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively. MALDI-MS/MS analysis detected only 3 TMP among 421 identified spots. However, when the samples had been washed with Na(2)CO(3), only few well-focused spots remained detectable on the gel covering the pH 6-11 range. Instead, a heavily ruthenium-stained smear became visible at the upper edge of the gel at the location where the samples had been applied by cup loading. LC-MS/MS analysis revealed that this smear contained 38 unfocused TMP with up to 12 transmembrane helices. After transfer to the second dimension, no major areas of protein staining were left on the IPG strips. This indicates that after extraction and denaturation the TMP may form high-molecular aggregates, due to their "hydrophobic interactions". These aggregates enter the IPG strips, but do not focus regularly. They are then transferred onto the 2-DE-gels, where they remain caught at the upper edge.

Technical strategies to reduce the amount of "false significant" results in quantitative proteomics.

When the p-value is set at <0.05 in statistical group comparisons, a 5% rate of "false significant" results is expected. In order to test the reliability of our 2-DE method, we loaded each of 24 gels with equal-sized samples (200 mug protein from pooled rat brain, pH 4-7, stained with ruthenium fluorescent stain for visualization) and statistically compared the first 12 gels with the last 12. In numerous experiments the rate of significant differences found far exceeded 5%. Several factors were identified as causing the following rates of false significant differences in spot intensities: (i) running samples in two different 2-DE runs (42%), (ii) running second dimension gels produced in two different gel casters (16%), (iii) normalizing the entire gel instead of separately normalizing several different gel zones (11%), (iv) using IPG strips from different packages (19%), (v) dividing the whole sample into subgroups during software analysis (9%). After controlling for all these factors, the rates of "false positive" results in our experiments were regularly reduced to approximately 5%. This is an indispensable prerequisite for avoiding too high a rate of false positive results in experiments in which different subgroups are compared statistically.

Improved comparative proteome analysis based on two-dimensional gel electrophoresis.

The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two-dimensional electrophoresis gels (pH 4-7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 microg and 250 microg protein, respectively. Only 72 spots were higher (p<0.05), while 58 spots were significantly lower in the 250-microg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between-gel variation. This resulted in the detection of significant differences in spot intensities in 77-90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.

Thyroid hormones in the rat amygdala as common targets for antidepressant drugs, mood stabilizers, and sleep deprivation.

BACKGROUND: There have been repeated reports of antidepressant effects of thyroid hormones. In this study, we investigated whether antidepressant treatments enhance the concentrations of thyroid hormones in rat brain. METHODS: Each of the groups of rats was treated for 14 days with one of the following: an antidepressant drug (desipramine, paroxetine, venlafaxine, or tianeptine); a mood stabilizer (lithium or carbamazepine); or 8 hours' partial sleep deprivation. Thyroid hormone concentrations were quantified in homogenates, nuclei, mitochondria, synaptosomes, myelin, and microsomes in 11 rat brain areas. RESULTS: No drug effects were seen on nuclear triiodothyronine (T(3)) concentrations in any brain area. In the amygdala, all antidepressant drugs enhanced the levels of T(3) in the myelin fraction. Triiodothyronine molecules were identified in the myelin by immunogold labeling. Quantification of the major lipid components showed a selective decrease in cholesterol in the myelin of the amygdala after desipramine treatment. Desipramine induced an increase in protein concentrations, 3,5-diiodothyronine levels, and the activity of the mitochondrial enzyme succinate dehydrogenase in the mitochondria of the amygdala. Lithium, carbamazepine, and partial sleep deprivation raised the levels of T(3) in synaptosomes of the amygdala. CONCLUSIONS: These results demonstrate that thyroid hormones in the amygdala are a common target of different antidepressant and mood-stabilizing therapies.

Effects of hyper- and hypothyroidism on thyroid hormone concentrations in regions of the rat brain.

The purpose of this study was to investigate the effects of hyper- and hypothyroidism on thyroid hormone concentrations and deiodinase activities in nine regions of the rat brain. Four weeks of treatment with 75 microg thyroxine (T4)/kg body wt induced a two- to threefold increase in T4 levels in all of these brain regions, whereas the 3,5,3'-triiodothyronine (T3) concentrations were reduced in five brain regions and remained unchanged in four. Even after 8 wk treatment with 300 microg T4/kg, the T3 concentrations remained normal in cortical areas, the hippocampus and amygdala, and were elevated only in areas in which inner-ring deiodinase activity was low or absent, and in the hypothalamus. At the subcellular level, nuclear concentrations of T3 were diminished in hypothyroidism but remained unaltered in hyperthyroidism in all areas except the hypothalamus, where they were enhanced. Cortical mitochondrial succinate dehydrogenase activity was reduced in both hypo- and hyperthyroidism in spite of normal T3 concentrations in hyperthyroid animals. The results show that nuclear T3 concentrations fall in hypothyroidism but do not change during severe hyperthyroidism in any brain region except the hypothalamus. Further research is thus needed to clarify the mechanisms mediating the numerous biochemical and psychological effects of hyperthyroidism.