CC BY
8[5]. Nitz DA, van Swinderen B, Tononi G, Greenspan RJ. Electrophysiological correlates of rest and activity in Drosophila melanogaster. Curr Biol, 2002, 12(22) : 1934-1940
[6]. Doronkin S, Reiter LT. Drosophila orthologues to human disease genes: an update on progress. Prog Nucleic Acid Res Mol Biol, 2008, 82 : 1-32
[7]. Bushey D, Cirelli C. From genetics to structure to function: exploring sleep in Drosophila. Int Rev Neurobiol, 2011, 99 : 213-244
[8]. Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C. Sleep homeostasis in Drosophila melanogaster. Sleep, 2004, 27(4) : 628-639
[9]. Harbison ST, Sehgal A. Quantitative genetic analysis of sleep in Drosophila melanogaster. Genetics, 2008, 178(4) : 2341-2360
Sleep-awakening Physiological Structure in Different Day-old Wild-typeCS
Drosophila
Li Tingli, Zhang Ji
(College of Pharmacy of Heilongjiang University of Chinese Medicine, Harbin, China)
Abstracts. Study Objectives: Fruit flies genetic advantage to promote sleep molecular mechanism research, and evaluate its sleep parameters are often just total sleep time, in order to better explore different phenotype fruit flies and wild type fruit flies sleep regulation process difference, we adopt large sample to determine 7 age male and female flies sleep - awakening physiological structure, more male and female flies sleep-awakening physiological structure of the difference. . Reveal different day age drosophila sleep - awakening physiological structure changes.
Design: Choose wild type Canton S fruit flies for experimental object, using fruit flies activity monitoring system (DAMS), respectively record total sleep time, sleep times, each time sleep duration, and drew the 24 hours of sleep structure and activity rhythm diagram to clarify drosophila sleep - awakening physiological structure.
Results: 1.7 days of age female fruit fly all day long on average sleep time is 606 minutes, and sleep times is 17 times, each time sleep duration is 47 minutes; 7 age male fruit fly all day long on average sleep time is 9 minutes, sleep times is 23 times, each time sleep duration is four minutes. 2. Female flies with age increase round-the-clock total sleep time gradually raised, gradually increase the number of sleep, every time sleep duration gradually reduced; Male fruit flies with age increase round-the-clock total sleep time no obvious change, sleep times increase gradually, every time sleep duration reduced gradually. 3. Along with the age increasing drosophila activity rhythm sex is not obvious, night and day, alternating activity peak disappear.
Conclusion: 7 age drosophila sleep - awakening physiological structure stability, rhythmic good, different day age wild type CS drosophila sleep-awakening physiological structure changes, along with the increase of day age drosophila sleep-awakening rhythm strength is reduced, sleep consolidate sex is abate, sleep fragmentation increased obviously.
Keywords: Drosophila, sleep - awakening physiological structure, Biological rhythm, DAMS
Introduction
In 2000, two independent lab published data show the fruit fly class sleep state characteristics. Although different approaches, but have found it surprising similarity: wild type fruit flies in the light and dark alternate and continuous dark two conditions male and female are for the performance of the night long time of fixed[1][2], opens the fruit fly as sleep medicine research model system of the new era. And evaluate its sleep parameters are often just total sleep time, has certain limitation, because one day of the sleep may be assigned to different time periods. If sleep
total time of the same gene mutations drosophila sleep structure changed, that the gene is likely to be about sleep mechanism. Therefore, determine the fruit fly within 24 hours of normal sleep -awakening physiological structure is very important. However, fruit flies life of sleep - of the awakening of the physiological structure is not more detailed clarify.
We consider gender and age two factors, on the fly a lot of experimental research. Foreign scholars often flies age is 5-10 days of age, through our laboratory study found that 7 age drosophila sleep of the awakening of the best state, so the experiments to large sample to determine 7 age drosophila sleep-awakening rhythm normal condition. At room temperature, black abdomen drosophila life approximate two months, in order to ensure enough sample size fruit fly, drosophila sleep monitoring for the biggest day age 50 days of age.
Materials and methods
Animals: wild-type Canton-S Drosophila melanogaster (College of Life Science, Peking University) . Food: H2O, cornmeal,sucrose,agar,dried live yeas,propionicD Flies that had been socially housedwere anaesthetised using CO2 and single fliestransferred to a 5 mmx665 mm polycarbonate tube containingfood. The tube was sealed at the food end withParafilm, withthe opposing end sealed with a cotton wool plug to allow for airtransfer. Fly sleep-wake behaviour was monitored using theDrosophila Activity Monitoring System (DAMS, TriKinetics,Waltham, MA, USA). All experimental procedures were carriedout at (24±1)oC in a humidified incubator. Flies were monitored on a 12 hr:12 hr light:dark cycle. All analysis wasdone with data collected from days 2 to 4 after flies were placedinto the DAMS. Sleep was regarded as a period of five minuteswithout beam crossing (as defined previously by other investigators. Data was collected using TriKinetics software and analysedin Excel (Microsoft).
Through the EXCEL software to process experimental data, drawing use time as abscissa, appear frequencies as ordinate. Drawing use day-old as abscissa, timeas ordinate. Drawing use day-old as abscissa, times of sleepas ordinate. Drawing use day-old as abscissa, average per sleepduration as ordinate. Drawing use time as abscissa, drosophila per hour of sleep time average as ordinate. Drawing use time as abscissa, average activities as ordinate. Using SPSS l3.0 analysis software, different types of data first through the normal distribution test, each data accord with normal distribution.
Results and discussion
As shown in the Figure 1 and 2, 7 age female flies total sleep time frequency in the 400-700 minutes is obviously higher than that of other range and 7 age male fruit flies total sleep time frequency in the 800-1100 minutes is obviously higher than that of other range. Although there are individual differences , but the total sleep time in line.
As shown in the Figure 3, 4, 5, 6 and 7, along with the age increase male and female flies sleep time have increase trend, little change in sleeptime at night. Female fly daytime sleep time increased and sleep time of male fliesis increased to reduceduring the day. Male and female fly sleep times increase gradually but every time sleep duration have downward trend, sleep fragmentation increase.
The Figure 8 and 9 shows that 7 days female drosophila's rhythm of day activity and night sleep is obvious; 30 days old female drosophila's day sleep is gradually increasing and night sleep is gradually reducing; the 50 days old female drosophila sleeps for half of their day, and night sleep is reduced; 7 days male drosophila sleeps in both day and night and sleep times equal, they are only awake when day and night alternate; 30 days male drosophila's sleep time in day and night has the trend of increasing, and they are not easy to awake when day and night alternate; 50 days old male drosophila's sleep time in day and night reduced again, especially reduced in night.
frequency o o c o o O h—1 ts o en i—'■ en to c "1 _i_ 0+ 60 0+ 700+ 800+ 900+ 1000+ 1100+ 1200+ 1300+ mins
Figure 1 Frequency histogram of 7 age female flies total sleep time
Figure 2 Frequency histogram of 7 age male flies total sleep time
female
male
1600 1400 1200 1000 800 600 400 200 0
-I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I_I
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
ages
Figure 3 Sleep time
The active rhythm of 7, 8, 9 days old drosophila is obvious, the day and night alternative activity peak is outstanding. 28, 29, 30 days old female drosophila has less day and night alternate activities. 48, 49, 50 days old female drosophila's active rhythm is not obvious and day and night activity peak disappeared. 28, 29, 30 days old male drosophila's day and night alternate activities reduced obviously. 48, 49, 50 days old male drosophila's active rhythm is not obvious, day and night alternate activity peak is disappeared, and activities in night increased. (Figure 10, 11)
Figure 4 Sleep time at day
—female —■—male
Figure 5 Sleep time at night
• female —"—male
ages
Figure 6 Sleep number
This study compares the different age sex fruit fly within 24 hours of sleep- awakening physiological structure, found that male and female flies daily sleep average duration no significant difference, with human similar, but male fruit flies total sleep time, sleep times are significantly higher than female fruit flies, activity rhythm also significantly different. Humans also have some differences between the sexes, Women's sleep spindle twice as much as men, have more slow wave sleep, delta wave in different time activities, along with the increase of age delta wave attenuation more slowly[3]. Many species of sleep mode is a bisexual binary, in the study found in cats, the male slow wave activities of the delta wave with the increase of age decay faster [4]. Male rats have more
167
REM sleep[5]. Different gender in mice in light and dark cycle a stage also showed REM sleep time different. In addition, in mice eat caffeine sleep structure also showed sexual dimorphism, male slow wave sleep rise and female fast wave sleep rise[7]. All of these results suggest that many different organisms, including human hormones affect sleep, sleep physiology and pathology of sleep. We found that male and female flies sleep structure is a bisexual binary, especially obvious difference, can study a bisexual sleep patterns of genetic mechanism.
-♦—female —■—male
ages
Figure 7 Average sleep bout length
Figure8Minutes of sleep per 30 minutes for female flies
oooooooooooooooooooooooo oooooooooooooooooooooooo
C^OOOTOI—lojco^Locot^aoOTOi—lojcooi— N m ^ ID CD
1—Il—Il—Il—Il—Il—Il—Il—Il—Il—IC^C^C^C^
time
» 7 days old —■—30 days old a 50 days old
Figure9Minutes of sleep per 30 minutes for male flies
Figure 10 Actograms showing averaged beam crossing data for three consecutive days for female flies
48 49 до
age(days)
day night day night day night
Figure 11 Actograms showing averaged beam crossing data for three consecutive days for male flies
kLiii Л i iikALu Л i ukALi J - iikiil
age (day s)
This study also compares the different with gender, age drosophila within 24 hours of sleep-awakening physiological structure, found that with the increase of day age drosophila sleep -awakening rhythm strength is reduced, increased daytime sleep, sleep in the night to reduce, sleep consolidate sex is abate, sleep fragmentation increased obviously. This phenomenon and human is very similar, as the growth of the age people sleep became not consolidated, on health are of great harm, and its potential mechanism still know very little[8]. Sleep-awakening is a kind of the most common one of the circadian rhythms, sleep disorders are likely to be diurnal rhythm disorders and biological clock function lack results, has been with the clock gene (PER, TIM, CLK) connected [9], and the gene in fruit flies first found in it, and so study age-related sleep - awakening rhythm has a lot of advantages.
With the maturity of gene technology, more and more mutant was applied in looking for sleep gene, believe that fruit flies this model organisms will be helping to unlock the mystery of sleep. Conclusions
we have found that sex affect the locomotor activity rhythms of drosophila of the same day-old, and the rhythms of the sleep-awakening declines with age. References
1. Hendficks JC, Finn SM, PanckefiKA, et a1., Best in drosophilais a sleeplike state. Neuron. 2000, 25(1): 129-138.
2. Shaw PJ, Cirolli C, Greenspan IL, et a1. Correlates of sleep andwaking in Drosophila melanogaster. Science, 2000, 287: 1834-1837.
3. McCarley RW. Neurobiology of REM and NREM sleep. Sleep Med, 2007, 8:302-30.
4. Bowersox SS, Floyd T, Dement WD. Electroencephalogram duringsleep in the cat: age effects on slow wave activity. Sleep, 1984, 7:380-385.
5. Fang J, Fishbein W. Sex differences in paradoxical sleep: Influencesof estrus cycle and ovariectomy. Brain Research, 1996, 734:275-285.
6. Bright PF, Fishbein W. Gender differences in the ultradian of sleep.Society for Neuroscience Abstracts, 1987, 13:264.
7. Sinton CW, Valatx JL, Jouvet M. Increased sleep time in the offspringof caffeine-treated dams from two inbred strains of mice.Neuroscience Letters, 1981, 24:169-174.
8. Koh K, Evans JM, Hendricks JC, Sehgal A: A Drosophila model for age-associated changes in sleep:wake cycles. Proc Natl Acad Sci USA, 2006, 103:13843-13847.
9. Ikehara S, Iso H, Date C, Kikuchi S, Watanabe Y, Wada Y, Inaba Y, Tamakoshi A: Association of sleep duration with mortality from cardiovascular disease and other causes for Japanese men and women: the JACC study. Sleep, 2009, 32: 295-301.
Ultra-performance LC-ESI/quadrupole-TOF MS for rapid analysis of chemical
constituents of Longdan-Xiegan decoction
*
YUJIA SUN,SHANSHAN WANG,HUI SUN, ZHIGANG WANG and XIJUN WANG
Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, National TCM Key Lab of Serum Pharmacochemistry, Heping road 24, Harbin, China;
Abstracts: The traditional Chinese medicine,Londan-Xiegan decoction(LXD),is a classic formulae containing ten medicinal herbs,Gentianae Radix,ScutellariaeRadix,Gardeniae Fructus,AlismatisRhizoma,Akebia Trifoliata Varaustralis,AngelicaeSinensisRadix,Rehmanniae Radix,Bupleuri Radix,GlycyrrhizaeRadix et Rhizoma,PlantaginisSemen.It is commonly used to treat jaundice,conjunctival congestion,earache,scrotum and extremitas inferior eczema,etc.Recently,we conducted in depth discussion and research in the modern clinical,made more achievements,to achieve further development and application of LXD has an important guiding significance.In this study,a sensitive and specific rapid ultra-performance LC-ESI/quadrupole-TOF high-definition MS (UPLC-ESI-Q-TOF-MS) with automated MetaboLynx analysis in negative ion mode were established to characterize the chemical constituents of LXD.The analysis was performed on a Waters UPLCTM HSS T3 (2.1 x100 mm,1.8 |m) using gradient elution system.MS/MS fragmentation behavior was proposed for aiding the structural identification of the constituents.By comparing the retention time and mass spectrometry data and retrieving the reference literatures,a total of 66 peaks were provisionally characterized with the optimization of conditions.Of note,5 ingredients were identified from Gentianae Radix,24 were from ScutellariaeRadix,8 were from Gardeniae Fructus,2 were from AlismatisRhizoma,5 were from Akebia Trifoliata Varaustralis,3 were from AngelicaeSinensisRadix,4 were from Rehmanniae Radix,4 were from Bupleuri Radix,7 were from GlycyrrhizaeRadix et Rhizoma,4 were from PlantaginisSemen.To concluded,UPLC-ESI-Q-TOF-MS,as a reliable and efficient platform,was successfully developed for globally identifying multiple-gradient of Chinese medicine prescriptions.
Keywords: Constituents;Traditional Chinese medicine;Identification;Longdan-Xiegan decoction;UPLC-ESI-Q-TOF-MS
1. Introduction
Traditional Chinese medicine(TCM) is the crystallization of the wisdom of the Chinese nation for thouthands of years,it not only has made an indelible contribution to the reproduction of both Chinese nation and people's health,but also greatly promoted a lot of neighboring Asian countries' medical career development[1].Due to their long historical clinical use and reliable therapeutic efficacy,the international influence of TCM is increasing day by day.It is indispensable to develop a rapid and effective analytical method to identify the constituents of TCM,then ensure the reliability in clinical application and increase quality control[2-8].
