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Hylak® forte is a postbiotic that inhibits the growth of pathogenic bacteria by reducing intestinal pH. It is assumed the potential presence of short-chain fatty acids (SCFAs) in Hylak® forte may contribute to this effect. In this current study, we analysed the composition of Hylak® forte, using a validated gas chromatography assay test method, to ascertain whether SCFAs are present in this postbiotic treatment. Hylak® forte was screened for C1 to C10 SCFAs by a gas chromatographic assay. In this assay, SCFAs were analysed as for their volatile ethyl ester derivatives in a 3.0 mL Hylak® forte sample. An additional screening procedure was conducted for the presence of vitamins and simple sugars. The gas chromatographic method for determining SCFAs was validated according to the requirements of ICH guideline Q2 (R1). Formic and acetic acids were identified in Hylak® forte at 27.92 ppm (90% confidence interval (CI), 26.90-28.94) and 306.17 ppm (90% CI, 277.11-335.22), respectively. Additional compounds were quantified in the solution, including vitamin B1 (0.029 mg/100 g), monosaccharides and disaccharides (2.767 g/100 g), as well as glutamic acid and glutamine (0.047 g/100 g). This study has identified formic acid in a range of 39.33 (90% CI, 36.50-42.17)-48.33 (90% CI, 45.91-50.76) ppm and acetic acid dropping down to 312.33 (90% CI, 295.32-329.35) from initial 415.67 ppm (90% CI, 385.93-445.41) in commercial Hylak® forte samples under forced degradation conditions. In addition, a range of other compounds in Hylak® forte was identified, including riboflavin and glutamine. Further studies are necessary to establish whether these compounds translate into tangible therapeutic effects.

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Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence elements. First, a selectable marker is PCR-amplified with synthetic primers attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary restriction site absent in the substrate plasmid. The resulting cassette is co-electroporated with substrate plasmids in Red/ET-proficient Escherichia coli cells. Following isolation of recombinant plasmids, linear nonselectable DNA replaces the cassette and introduces the desired mutation(s) in a second Red/ET recombination step. Upon selective digestion of parental plasmids and retransformation, a 38% mutation efficiency was achieved using a synthetic 97-nucleotide oligonucleotide to cure a 17-bp deletion within lacZalpha of pUC19 (2,686 bp). A PCR fragment was used with similar efficiency to co-replace mouse Cdkn1b codons 9 and 76 in gene-targeting vector pGTC (13,083 bp).

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The circadian clock has been implicated in addiction and several forms of depression [1, 2], indicating interactions between the circadian and the reward systems in the brain [3-5]. Rewards such as food, sex, and drugs influence this system in part by modulating dopamine neurotransmission in the mesolimbic dopamine reward circuit, including the ventral tegmental area (VTA) and the ventral striatum (NAc). Hence, changes in dopamine levels in these brain areas are proposed to influence mood in humans and mice [6-10]. To establish a molecular link between the circadian-clock mechanism and dopamine metabolism, we analyzed the murine promoters of genes encoding key enzymes important in dopamine metabolism. We find that transcription of the monoamine oxidase A (Maoa) promoter is regulated by the clock components BMAL1, NPAS2, and PER2. A mutation in the clock gene Per2 in mice leads to reduced expression and activity of MAOA in the mesolimbic dopaminergic system. Furthermore, we observe increased levels of dopamine and altered neuronal activity in the striatum, and these results probably lead to behavioral alterations observed in Per2 mutant mice in despair-based tests. These findings suggest a role of circadian-clock components in dopamine metabolism highlighting a role of the clock in regulating mood-related behaviors.

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Over many years evidence accumulated that circadian rhythms are related to psychiatric disorders.1-3 However, a mechanistic relationship between the circadian clock and mood related behaviors remained enigmatic. Now, we have reported that monoamine oxidase A (MAOA), a mitochondrial enzyme degrading catecholamines including dopamine, is regulated by components of the circadian clock.4 Interestingly, this regulation is variable depending on cell type, indicating the presence of cell type specific factors modulating BMAL1/NPAS2 or BMAL1/CLOCK dependent transcription. In the mesolimbic dopaminergic reward circuit, including the ventral tegmental area (VTA) and the ventral striatum/nucleus accumbens (NAc) we found a positive influence of PERIOD 2 (PER2) on transcriptional activation of Maoa using mice mutant in the Per2 gene. These animals show less Maoa mRNA expression and MAO activity compared to wild type littermates. This is probably the reason for the observed increase in dopamine levels in the striatum of Per2 mutant mice what leads to alteration in despair-based behavioral tests. These results suggest that clock components can influence dopamine metabolism and mood-related behaviors.

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Most behavioral experiments within circadian research are based on the analysis of locomotor activity. This paper introduces scientists to chronobiology by explaining the basic terminology used within the field. Furthermore, it aims to assist in designing, carrying out, and evaluating wheel-running experiments with rodents, particularly mice. Since light is an easily applicable stimulus that provokes strong effects on clock phase, the paper focuses on the application of different lighting conditions.