Luciferase reporter assays represent a simple and sensitive experimental system in cell and molecular biology to study multiple biological processes. However, the application of these assays is often limited by the costs of conventional luminometer instruments and the versatility of their use in different experimental conditions. Therefore, we aimed to develop a small, affordable luminometer allowing continuous measurement of luciferase activity, designed for inclusion into various kinds of tissue culture incubators. Here, we introduce LuminoCell-an open-source platform for the construction of an affordable, sensitive, and portable luminometer capable of real-time monitoring in-cell luciferase activity. The LuminoCell costs $40, requires less than 1 h to assemble, and it is capable of performing real-time sensitive detection of both magnitude and duration of the activity of major signalling pathways in cell cultures, including receptor tyrosine kinases (EGF and FGF), WNT/β-catenin, and NF-κB. In addition, we show that the LuminoCell is suitable to be used in cytotoxicity assays as well as for monitoring periodic circadian gene expression.
OBJECTIVE: To evaluate changes in the expression of clock genes and melatonin levels in patients with idiopathic REM sleep behavior disorder (RBD) as a potential early stage of synucleinopathies. METHODS: We assessed the rhythmicity of circadian clock genes using real time-quantitative polymerase chain reaction and 24-h blood melatonin profiles using radio-immunoassay in 10 RBD patients and nine age-matched controls. RESULTS: The RBD patients did not show circadian rhythmicity for clock genes Per2, Bmal1, and Nr1d1 but the rhythmicity of Per 1 remained, and the amplitude of Per3 was diminished. The 24-h melatonin rhythm did not differ between RBD patients and healthy control subjects. Melatonin profile in RBD patients was delayed by 2 h compared to controls, the habitual sleep phases were phase delayed by about 1 h, however no phase shift occurred in any of the clock genes studied. The control group had stable acrophases of melatonin rhythms of approximately 5 h whereas the RBD patients had a more dispersed range over 11 h. CONCLUSIONS: Our results suggest that RBD could be associated with altered expression of clock genes and delayed melatonin secretion. Thus, we argue that circadian system dysregulation could play a role in RBD.
- MeSH
- cirkadiánní proteiny Period genetika MeSH
- cirkadiánní rytmus genetika MeSH
- exprese genu * MeSH
- jaderné receptory - podrodina 1, skupina D, člen 1 genetika MeSH
- lidé MeSH
- melatonin krev metabolismus MeSH
- polysomnografie MeSH
- porucha chování v REM spánku genetika MeSH
- proteiny CLOCK genetika MeSH
- průzkumy a dotazníky MeSH
- senioři MeSH
- stadia spánku genetika MeSH
- transkripční faktory ARNTL genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- hodinové geny,
- MeSH
- biologické hodiny fyziologie genetika MeSH
- bipolární porucha farmakoterapie MeSH
- chlorid lithný farmakologie terapeutické užití MeSH
- cirkadiánní proteiny Period genetika MeSH
- cirkadiánní rytmus * fyziologie genetika MeSH
- depresivní poruchy farmakoterapie MeSH
- epigenomika MeSH
- ketamin farmakologie terapeutické užití MeSH
- lidé MeSH
- melatonin metabolismus MeSH
- mikro RNA MeSH
- poruchy cirkadiánního rytmu (spánek) MeSH
- poruchy nálady * etiologie farmakoterapie MeSH
- proteiny CLOCK genetika MeSH
- schizofrenie * etiologie farmakoterapie MeSH
- sliny chemie metabolismus MeSH
- spánková deprivace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Alzheimer's disease (AD) is a neurodegenerative disease often accompanied with disruption of sleep-wake cycle. The sleep-wake cycle is controlled by mechanisms involving internal timekeeping (circadian) regulation. The aim of our present pilot study was to assess the circadian system in patients with mild form of AD in their home environment. In the study, 13 elderly AD patients and 13 age-matched healthy control subjects (the patient's spouses) were enrolled. Sleep was recorded for 21 days by sleep diaries in all participants and checked by actigraphy in 4 of the AD patient/control couples. The samples of saliva and buccal mucosa were collected every 4 hours during the same 24 h-interval to detect melatonin and clock gene (PER1 and BMAL1) mRNA levels, respectively. The AD patients exhibited significantly longer inactivity interval during the 24 h and significantly higher number of daytime naps than controls. Daily profiles of melatonin levels exhibited circadian rhythms in both groups. Compared with controls, decline in amplitude of the melatonin rhythm in AD patients was not significant, however, in AD patients more melatonin profiles were dampened or had atypical waveforms. The clock genes PER1 and BMAL1 were expressed rhythmically with high amplitudes in both groups and no significant differences in phases between both groups were detected. Our results suggest moderate differences in functional state of the circadian system in patients with mild form of AD compared with healthy controls which are present in conditions of their home dwelling.
- MeSH
- aktigrafie MeSH
- Alzheimerova nemoc komplikace patofyziologie MeSH
- bydlení MeSH
- chorobopisy MeSH
- cirkadiánní proteiny Period biosyntéza genetika MeSH
- cirkadiánní rytmus fyziologie MeSH
- lidé MeSH
- melatonin analýza MeSH
- messenger RNA analýza biosyntéza MeSH
- poruchy spánku z vnitřních příčin komplikace patofyziologie MeSH
- regulace genové exprese MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- sliny chemie MeSH
- studie případů a kontrol MeSH
- transkripční faktory ARNTL biosyntéza genetika MeSH
- ústní sliznice chemie MeSH
- životní prostředí MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
