Shuting Cheng

Behavioral change related to Wenchuan devastating earthquake in mice

It has been suggested that some animals are much more capable of perceiving certain kinds of geophysical stimuli which may precede earthquakes than humans, but the anecdotal phenomena or stories about unusual animal behaviors prior to an earthquake should be interpreted with objective data. During the Wenchuan magnitude 8.0 earthquake that happened in Wenchuan county (31.0 degrees north latitude, 103.4 degrees east longitude) of Sichuan province, China, on May 12, 2008, eight mice were monitored for locomotor activity and circadian rhythm in constant darkness with temperature 22-24 degrees C and humidity 55-65% for 38 days. The ongoing monitoring of locomotor activity of mice in our laboratory made it possible to design a posteriori study investigating whether the earthquake was associated with any change in animal behavior. Based on analyzing the recorded data with single cosinor, we found that the locomotor activity dramatically decreased in six of these eight mice on day 3 before the earthquake, and the circadian rhythm of their locomotor activity was no longer detected. The behavioral change lasted for 6 days before the locomotor activity returned to its original state. Analyses of concurrent geomagnetic data showed a higher total intensity during the span when the circadian rhythm in locomotor activity weakened. These results indicated that the behaviors, including circadian rhythm and activity, in these mice indeed changed prior to the earthquake, and the behavioral change might be associated with a change of geomagnetic intensity.

The noncircadian function of the circadian Clock gene in the regulation of male fertility.

Mice homozygous for a dominant-negative allele of the Clock gene (ClockΔ19/Δ19) have slightly but significantly decreased male fertility. The molecular mechanism for this reduction in fertility is unknown. In the present study, we used a small hairpin RNA (shRNA) strategy to specifically knock down the Clock gene expression in the testes of male mice and determined its effect on male fertility. Clock knockdown led to smaller litter size, a lower in vitro fertility rate, lower blastula formation rate, and lower acrosin activity of the knockdown sperm. Locomotor activity analysis of the Clock knockdown mice revealed that Clock knockdown in testes did not alter their circadian rhythm. Taken together, these results provide the first evidence that Clock gene expression in round spermatids is essential for maintaining male reproductivity and suggest that acrosin may be a novel regulatory target of the Clock gene that would regulate the fertilization and early embryonic development to blastula. These findings may provide new clues for development of novel male contraceptive strategies.

Influence of the core circadian gene "Clock" on obesity and leptin resistance in mice.

Alterations in metabolism could be due to cell-autonomous effects associated with altered expression of Clock in central nervous system feeding centers and/or peripheral tissues involved in metabolism. Clock mutant mice are hyperphagic and obese, which indicates that Clock is related to obesity. In the present study, we used intracerebroventricular injection of recombinant adenoviral vector harboring Clock genes to explore the role of Clock on diet induced obesity and the mechanisms involved in leptin resistance and leptin signaling in mice. The results demonstrated that expression of Clock in the arcuate nucleus of diet induced obesity mice was down-regulated. The recombinant adenoviral vector harboring Clock genes could reduce obesity indexes of diet induced obesity mice including body weight, BMI and total fat mass, attenuate hyperleptinemia, increase leptin sensitivity and decrease accumulated suppressor of cytokine signaling-3 in the arcuate nucleus. These results indicate that Clock plays an important role on obesity, which may be involved in leptin resistance and regulation of suppressor of cytokine signaling-3 in arcuate nucleus.

Identification and characterization of the free fatty acid receptor 2 (FFA2) and a novel functional FFA2-like receptor (FFA2L) for short-chain fatty acids in pigs: Evidence for the existence of a duplicated FFA2 gene (FFA2L) in some mammalian species

Free fatty acid receptor 2 (FFA2, also called GPR43) is reported to play a critical role in mediating the actions of short-chain fatty acids (SCFAs) in humans and mice. However, little is known about the structure, functionality, and tissue expression of FFA2 in other mammalian species, including pigs. In the present study, the full-length cDNAs of FFA2 (pFFA2) and a novel FFA2-like gene (named pFFA2L) were cloned from pig intestines by reverse transcription PCR. Both cloned pFFA2 and pFFA2L are predicted to encode 2 receptors of remarkable structural similarity and share high amino acid sequence identities with FFA2 from other mammalian species. Interestingly, the novel FFA2L could also be identified in 9 other mammalian species, suggesting that FFA2L was likely duplicated from FFA2 in the last common ancestor of these species. With the use of a pGL4-SRE-luciferase reporter assay, we demonstrated that pFFA2 expressed in human embryonic kidney 293 cells could be activated by acetate, propionate, and butyrate equipotently, whereas pFFA2L could be activated only by acetate and propionate, indicating that both pFFA2 and pFFA2L are functional receptors for SCFAs with nonidentical pharmacologic properties. Reverse transcription PCR found that pFFA2 mRNA was widely expressed in nearly all tissues examined, including adipose tissue and gastrointestinal (GI) tract, whereas pFFA2L expression was mainly restricted to the GI tract. Taken together, our findings raise a novel concept that the actions of SCFAs are likely mediated by 2 FFA2s (FFA2 and FFA2L) in target tissues of some mammalian species, such as the GI tract of pigs.

An investigation of circadian rhythm in Escherichia coli

Background: Persuasive evidence for circadian programs in non-photosynthetic bacteria other than cyanobacteria is still lacking, we aimed to investigate the circadian rhythm of specific growth rate in Escherichia coli ATCC 25922, one of the important prokaryotes. Methods: To grow E. coli under different light and dark conditions. When the growth entered into the stationary phase, we stopped the culture and obtained the viable counts by MTT assay every 3 h. The specific growth rates (SGRs) were calculated and analyzed with cosinor method for potential rhythms. Results: Single cosinor method revealed that the SGR of E. coli displayed rhythmic variations with a period of around 24 h both under light/dark cycles and under constant darkness. The best-fitting periods and best-fitting cosine curves were acquired. Conclusions: The SGR of E. coli (ATCC 25922) in a culture medium with limiting substrates in the stationary and death phases displayed rhythmic variations with a period of around 24 h under light/dark cycles and constant darkness conditions.

Patterns of mortality from cardiac-cerebral vascular disease and influences from the cosmos

The aim of this study is to determine the patterns of mortality from cardiac-cerebral vascular disease (CCVD) and how they relate to cosmophysical factors over a six-year span. The daily number of deaths recorded in Chengdu over 2192 consecutive days (2007–2012), together with several factors related to solar activity (SA), geomagnetic activity (GMA), and the interplanetary magnetic field, were analyzed by the extended cosinor to estimate the parameters of major cycles characterizing each variable. Estimates of their respective periods with a measure of uncertainty serve to find coperiodisms (i.e. shared rhythms). The results show that mortality of cardiovascular diseases (CVD) and CCVD on days of GMA in quiet level was relatively higher on days of GMA in unsettled level. Whereas mortality data are characterized by a one-year synchronized component, most GMA and SA indices have periods differing from precisely one year. A half-year cycle is shared by almost all SA, GMA, and mortality variables with 95% confidence interval of the period covering 0.5 year. A transyear cycle (about 1.3 years) is found for all mortality data and many of the environmental variables. A cis-half-year (about 0.43 year) is shared between mortality from CCVD and flow pressure, sunspot numbers and storm-time variation. These results suggest the presence of complex temporal relations between patterns of CCVD mortality and cosmophysical activity.

Downregulation of Clock in circulatory system leads to an enhancement of fibrinolysis in mice

As a main component of circadian genes, clock plays not only an important role in circadian rhythm but also in the regulation of many physiological systems. The dysfunction of clock genes is associated with the development of various disorders. Many studies have investigated the association between clock genes and blood coagulation and the fibrinolytic system. The present study was designed to investigate the effect of downregulation of circulatory Clock on blood coagulation and fibrinolysis at the initial stage of active phase in male mice. Downregulation of the expression of the Clock gene by siRNA and, subsequently, its effect on the thrombotic potential and the expression of relative coagulative and/or fibrinolytic factors were investigated. It was found that the Clock interfered mice were less liable to thrombosis and showed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) at Zeitgeber time (ZT) 15. Meanwhile, these mice also showed an increase in factor VII (FVII) and a decrease in thrombomodulin (TM) and plasminogen activator inhibitor 1 (PAI-1) at ZT 15 at both transcriptional and translational levels. PT, APTT and mRNA expressions of fvii, tm and pai-1 were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method; no circadian rhythm was determined in PT and APTT, and a higher amplitude of fvii in the Clock RNAi group was found with a circadian phase shift, while lower amplitudes of tm and pai-1 were found in the Clock RNAi group with nearly no phase shift. All these results suggest that downregulation of the Clock gene in circulatory system has an effect on factors involved in both blood coagulation and fibrinolysis resulting in an enhancement in mice. This may be considered as an indication that Clock regulates thrombotic homeostasis through the fibrinolytic system.

The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements.

Nicotinamide adenine dinucleotide: a possible circadian zeitgeber functioning in nontranscription oscillation

Emerging evidences have shown that circadian rhythm can also be regulated by a nontranscriptional mechanism. In this study, we investigated the in vitro rhythmic characters of peroxiredoxin2 (PRX2) and nicotinamide adenine dinucleotide (NAD) in red blood cells (RBCs) and/or in heparin anticoagulated plasma under different light conditions. Our results showed that NAD had a free-running rhythm in accordance with that of PRX2 in RBCs, and both rhythms were liable to light entrainment. A decreased expression of PRX2 in RBCs and a notable phase delay of locomotor activity were found in mice intravenously injected with NAD, suggesting that NAD in nontranscriptional oscillating systems could receive the light zeitgeber signal and influence the expression of PRX2. NAD may also act as a nonphotic zeitgeber, which could make an advanced phase shift of per2, entraining the transcriptional oscillating system to make these two oscillating systems synchronized in a very short time. This zeitgeber might be of great significance as a mediator between these two oscillating systems.

The circadian intracranial pressure would be a prognostic factor in traumatic brain injury

Abstract Traumatic brain injury (TBI) generally influences circadian rhythms and has been implicated changes in circadian rhythm. Whether TBI-induced changes in circadian rhythm may affect the prognosis or recovery from TBI remains to be investigated. Sixty-two patients with TBI were continuously monitored for intracranial pressure (ICP) during the first 24 hours after the implantation of ICP monitor. The data from each patient were analyzed using the least squares fit of a 24-h cosine function by single cosinor method. Parameters of circadian A (Amplitude)/M (MESOR) were used to evaluate the circadian rhythm of the patients. Student’s t-test and Pearson’s chi-squared test were utilized to analyze the differences between good prognosis group and poor prognosis. A linear regression analysis was then applied to calculate the correlation between circadian A/M of ICP and Glasgow Coma Scale (GCS) before discharge, the Extended Glasgow Outcome Scale (GOS-E), the dosage of mannitol, and time spent in the intensive care unit (ICU), respectively. The results demonstrated that circadian A/M of patients’ ICP exhibited a positive correlation with GCS scores taken before discharge, GOS-E scores, and was negatively correlated with the amount of mannitol, and time spent in the ICU. We conclude that changes in the ICP circadian rhythm in TBI patients could reflect an internal signal of brain damage and, therefore, may be useful to predict a patient’s prognosis and recovery from TBI.