Background: In the present study, we explored the possibility of the stability of attention bias and memory bias in currently remitted individuals with unipolar depression compared to currently depressed individuals with unipolar depression and never-depressed individuals. Methods: The Emotional Stroop and autobiographical memory task (AMT) were administered on 10 participants, who were currently depressed, currently remitted with unipolar depression, or never-depressed. In the emotional Stroop task (EST), the respondent’s task was to indicate the color of the ink of the positive, negative, and neutral words by selecting one of a series of colored blocks. In the AMT, participants were presented with positive, negative, and neutral cue words. For each word, they were asked to report specific events from their life. Results: Both the attention bias and memory bias exist in both the clinical groups. In EST, both currently depressed and currently remitted groups were slower to respond to negative words compared to neutral words. Unlike EST, in AMT both currently depressed and currently remitted groups were slower to respond to positive words compared to neutral words. Interestingly, the capacity to generate specific events for negative events was higher in both currently depressed and currently remitted groups. They were over-general in their memories of positive events. Importantly, the never-depressed group was specific in their memories of both positive and negative events of their life. Conclusion: Our findings provide evidence for the stable existence of attention and memory bias in currently remitted individuals. This study has implications for the cognitive behavior therapy for depression to include modules to resolve the attention and memory bias toward negative thought and content, and to build strategies to overcome such biases. Cognitive processes like selective attention and memory influence the affective states and vice versa. Greater allocation of these cognitive processes to a particular category of stimuli is referred to as cognitive bias. The question of cognitive biases such as attention bias and memory bias has long held interest in the study of cognitive mechanisms underlying depression. These biases are critical aspects of several cognitive theories of depression Beck, . These tThe cognitive theory also postulates cognitive biases as predisposing or susceptibility factors to depression. Cognitive biases persist beyond the depressive state, which in turn may cause the relapse of depressive state. Consistent with Beck’s  theory, Gilboa and Gotlib  randomlyIt is also suggested that depressed individuals not only show memory bias in terms of the speed with which they can remember positive and negative events from their past, but they also find it difficult to be specific in their recall of events. This tendency has been found to be more evident with positive than negative events  for memory bias were used with three groups of participants. Interestingly, it should be noted that as yet only one study has looked at the processing of autobiographical memory style in currently remitted individuals with unipolar depression , currently remitted individuals with unipolar depression , and never-depressed individuals . All participants were between 21 and 53 years of age and their primary language was Hindi. Both clinical groups were recruited through the Department of Psychiatry, Swaroop Rani Medical College and Hospital in Allahabad city. All participants were invited for a Structured Clinical Interview for DSM-IV  primary diagnosis of major depression according to Diagnostic and Statistical Manual of Mental Disorders  of red, blue, and green respectively which appeared simultaneously on the screen . At the same time a white rectangular block (5.0 cm × 1.5 cm) which contained a word (1.00 cm high) appeared above the three colored blocks . There were 180 such trials, preceded by 30 practice trials.Memory bias: autobiographical memory task. The AMT  × 3 mixed factor design, where Group was a between group factor and word valence was a within group factor. In general, clinical groups showed more variability in their response compared to never-depressed group.F = 265.7, p < 0.001, and from the never-depressed group, F = 386.48, p < 0.001, on severity of depression. The currently remitted group with unipolar depression were not significantly different from the never-depressed group on the HDRS, suggesting that the clinical condition of these participants did not qualify for a diagnosis of major depression at the time of assessment and that they were symptom free  = 6.872, p < 0.01, with larger latencies for the currently depressed (M = 2.08 s), F = 4.8, p < 0.001, and currently remitted groups (M = 1.90 s), F = 4.08, p < 0.01, as compared to never-depressed group (M = 0.77 s). There was a significant interaction effect between group × word valence, F = 4.54, p < 0.01.The two-way ANOVA revealed a significant main effect of group, Post hoc analysis indicated that both the currently depressed, t = 2.27, p < 0.05, and currently remitted groups, t = 3.39, p < 0.01, were significantly slower to respond for negative words compared to neutral words. There was no significant difference in reaction time to respond for negative and neutral words in the never-depressed group, t = 0.192, p = 0.85. There was no significant difference in reaction time to respond for positive words compared to neutral words in the currently depressed, currently remitted, or never-depressed group . These results indicated that both the clinical groups showed attention bias to respond for negative words  = 11.26, p < 0.001, with significantly higher response latencies for positive words(M = 13.43 s) when compared to both negative (M = 10.67 s), F = 4.2, p < 0.01, and neutral (M = 9.63 s), F = 5.6, p < 0.001 cue words. There was a significant interaction effect between group and word valence, F = 12.82, p < 0.001.The two-way ANOVA revealed a significant main effect of word valence, Post hoc comparisons indicated that both the currently depressed, t = 4.91, p < 0.001, and currently remitted groups, t = 2.82, p < 0.05, were significantly slower to respond for positive cue words compared to neutral cue words. In contrast, the never-depressed group were slower to respond to negative cue words than neutral, t = 6.25, p < 0.001. There was no significant difference in reaction time to respond for negative cue words compared to neutral cue words in the currently depressed group, t = 0.120, p = 0.90. Interestingly, the currently remitted group responded faster for negative words compared to neutral words, t = 2.93, p < 0.01. There was no significant difference in reaction time between positive and neutral cue words in never-depressed group, t = 0.856, p = 0.41. These results indicated that negative bias for recalling events from memory was found in both the currently depresses and currently remitted groups, and interestingly both clinical groups appear to have lost the positive memory bias that characterized never-depressed group  = 9.50, p < 0.001, with memories of both the currently depressed group (M = 53.3%), F = 5.3, p < 0.001, and the currently remitted group (M = 53.3%), F = 5.3, p < 0.001, being less specific than those of never-depressed group (M = 70%). The main effect of cue valence was also significant, F = 106.82, p < 0.001, with less specific responses to positive, F = 11.0, p < 0.001, and neutral cue words, F = 20.6, p < 0.001, compared to negative cue words. Memory specificity was highest for negative cue words (M = 83.3%) followed by positive cue words (M = 57.7%) with worst specificity for neutral cue words (M = 35.5%). Interestingly, there was a significant interaction between group × cue valance, F = 16.15, p < 0.001.The two-way ANOVA revealed a significant main effect of group, Post hoc analysis indicated that both the currently depressed group, t = 9.79, p < 0.001, and the currently remitted group, t = 8.5, p < 0.001, were more specific when recalling negative events than neutral. There was no significant difference in memory specificity between positive and neutral memory for either the currently depressed, t = 1.50, p = 0.168, or currently remitted groups, t = 1.5, p = 0.16. Interestingly, the never-depressed group was more specific in both positive, t = 9.79, p < 0.001, and negative memories, t = 8.57, p < 0.001, compared to neutral events  then it gives an indication that both attention and memory biases are stable markers of depression.Confirming our prediction, we found that attention and memory bias both exist in both clinical groups. We found that both currently depressed and currently remitted individuals with unipolar depression were slower to respond to negative words in the EST and slower as well as less specific in recalling positive episodic events of their life. These results indicate that both currently depressed and currently remitted individuals selectively attended to the negative information even though that was not related to the particular task at hand. In addition, both clinical groups appear to have lost the positive memory bias that characterized never-depressed individuals. These findings indicate that like currently depressed individuals, currently remitted individuals with unipolar depression might also not be able to allocate sufficient resources to cognize positive information. This result is consistent with a previous report that currently remitted individuals with unipolar depression were more specific in their recall for negative events compared to currently remitted individuals with bipolar depression . Recent findings suggest that over-general memory may be reduced by cognitive interventions and maintained by ruminations (Watkins et al., It should be noted that from the current study, we cannot comment on whether abnormalities in both biases suggest underlying dysfunction in common brain systems or multiple brain systems. However, recently Disner et al.  integratPresent findings could have implications for treatment of depression. It appears that cognitive biases are more stable than was previously believed, and that these biases should be targeted through long-term specific interventions during, and even after, a depressive episode. Successful treatment of depression not only requires a successful resolution of depressed symptoms but, more importantly, change in the underlying, stable cognitive factors that increase susceptibility for repeated episodes. There are some reports which suggest that psychological treatment like cognitive behavioral therapy influence the later stage of information-processing, presumably via an effect on frontal control regions (Browning et al., In conclusion, the present study is the first study which clearly indicates that both attention and memory bias simultaneously exist beyond the depressive episodes, which suggests that both cognitive biases are stable markers of depression. Further studies could be done to see the relationship between the two biases to understand the etiology of depression. There were some limitations of the present study. The sample size in the present study was small and restricts the wider generalization of the results; thus this should be considered as a preliminary study showing promising trends about stability of cognitive biases. In addition the GHQ was administered only on the never-depressed group, which was an experimenter error. However, clinical history shows that both the clinical groups did not show any general health problems. Certainly, a replication of the present results with a larger sample and with more stringent selection criteria taking medication into account is warranted.Despite this limitation, our findings provide evidence of stability of attention and memory bias in currently remitted individuals with unipolar depression. This study has implications for future longitudinal studies, which could investigate whether the stability of both attention and memory biases in currently remitted individuals is predictive of relapse in unipolar depression. In addition, a longitudinal study will be helpful to explain the process as to how biases resolve from the state of severe depression through remission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The cortical silent period (CSP) elicited by transcranial magnetic stimulation (TMS) is affected by changes in TMS intensity. Some studies have shown that CSP is shortened or prolonged by short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF), Those studies, however, used different TMS intensities to adjust the amplitude of the motor evoked potential (MEP). Therefore, it is unclear whether changes in CSP duration are induced by changes in TMS intensities or by SICI and ICF. The purpose of this study was to confirm the effects of muscle contractions and stimulus intensities on MEP amplitude and the duration of CSP induced by single-pulse TMS and to clarify the effects of SICI and ICF on CSP duration.MEP evoked by TMS was detected from the right first dorsal interosseous muscle in 15 healthy subjects. First, MEP and CSP were induced by single-pulse TMS with an intensity of 100% active motor threshold (AMT) at four muscle contraction levels . Next, MEP and CSP were induced by seven TMS intensities  during muscle contraction of 10% EMG. Finally, SICI and ICF were recorded at the four muscle contraction levels .MEP amplitudes increased with increases in muscle contraction and stimulus intensity. However, CSP duration did not differ at different muscle contraction levels and was prolonged with increases in stimulus intensity. CSP was shortened with SICI compared with CSP induced by single-pulse TMS and with ICF at all muscle contraction levels, whereas CSP duration was not significantly changed with ICF.We confirmed that CSP duration is affected by TMS intensity but not by the muscle contraction level. This study demonstrated that CSP is shortened with SICI, but it is not altered with ICF. These results indicate that after SICI, CSP duration is affected by the activity of inhibitory intermediate neurons that are activated by the conditioning SICI stimulus. Motor evoked potential (MEP) is recorded from peripheral muscles after stimulation of the primary motor cortex by transcranial magnetic stimulation (TMS). TMS produces MEP followed by a period of electromyogram (EMG) silence during voluntary muscle contraction. This period of silence is known as the cortical silent period (CSP). While spinal mechanisms may be active in the early part (approximately 50 ms) of CSP, the cortical origin of at least the later part (approximately 100 ms) of CSP has been proven in several previous studies -8, and tRecently, paired-pulse TMS has become a useful tool for testing cortical inhibition or facilitation of the human motor cortex. When a subthreshold conditioning pulse (S1) and a suprathreshold test pulse (S2) were applied to the motor cortex through the same coil, MEP evoked by the test pulse was inhibited at interstimulus intervals (ISI) between 1 and 5 ms . This phThere have been some studies concerning CSP duration after SICI or ICF during muscle contraction -24, wherThe purpose of this study was to confirm the effects of muscle contractions and stimulus intensities on MEP amplitude and the duration of CSP induced by single-pulse TMS and to clarify the effects of SICI and ICF on CSP duration.The 1 mV TMS intensity and active motor threshold (AMT) were 56.5 ± 7.5% and 36.0 ± 4.7% [mean ± standard deviation (SD)], respectively, of the maximum stimulator output. Figure  = 45.758, p < 0.01; Figure Results of one-way analysis of variance (ANOVA) showed that MEP amplitude changed significantly with increased muscle contraction  = 0.466, p > 0.05; Figure CSP duration [mean ± standard error of the mean (SEM)] was 56.3 ± 1.0 ms at 10% EMG, 55.5 ± 1.6 ms at 30% EMG, 55.0 ± 2.1 ms at 50% EMG, and 54.8 ± 1.6 ms at 70% EMG. No significant differences in CSP duration were observed among the muscle contraction levels  = 67.687, p < 0.01; Figure Results of one-way ANOVA showed that MEP amplitude changed significantly with increases in TMS intensity  = 119.578, p < 0.01; Figure Results of one-way ANOVA showed that CSP duration changed significantly with increases in TMS intensity  = 18.712, p < 0.01; 10% EMG, F = 12.263, p < 0.01; 30% EMG, F = 21.738, p < 0.01; 50% EMG, F = 6.788, p < 0.05). The mean (± SEM) MEP amplitude at rest was 0.90 ± 0.10 mV (single-pulse TMS), 0.51 ± 0.08 mV (SICI), and 1.25 ± 0.18 mV (ICF). The MEP amplitude induced by SICI was significantly smaller than that induced by single-pulse TMS (p < 0.01). Furthermore, the MEP amplitude induced by ICF was significantly larger than that induced by single-pulse TMS (p < 0.05)  conditioning pulse intensity was 32.5 ± 5.8%, and the test pulse intensity was 56.5 ± 7.5%. Figure  = 27.744, p < 0.01; increase ratios, F = 11.571, p < 0.01; Figure Results of one-way ANOVA showed that the decrease and increase ratios changed significantly with increases in muscle contractions  = 12.734, p < 0.01; 30% EMG, F = 15.789, p < 0.01; 50% EMG, F = 24.969, p < 0.01; Figure Results of one-way ANOVA showed that CSP duration changed significantly with paired-pulse TMS  participated in this study. All subjects gave their written informed consent. This study was approved by the ethics committee at the Niigata University of Health and Welfare, Niigata, Japan (17279–111012).The subjects were seated comfortably in a chair. EMGs were recorded from the right FDI muscle using a silver/silver-chloride electrode in a belly tendon montage. EMG signals were amplified (×1000) by an amplifier  and digitized at 2 kHz using a A/D converter . The subjects performed right index finger abduction, and MVC and maximum EMG values of FDI were measured (100% EMG). The EMG signals were rectified and smoothed with a 501-point smoothing. The muscle contraction level was controlled by displaying a constantly updated bar graph showing the smoothed EMG amplitude . These monitors also allowed us to confirm the state of muscle contraction. The subjects were instructed to contract and sustain the targeted levels at 0 (rest), 10, 30, 50, and 70% EMG.Monophasic pulse TMS was delivered with a figure-of-eight-shaped coil  connected to Magstim 200 . The coil was held with the handle pointing backward and laterally at approximately 45° to the sagittal plane. The optimal spot to elicit MEP in the right FDI was carefully determined in each subject, and the optimal coil position to evoke a stable MEP was marked on the cap worn by the subject. AMT was obtained during a slight isometric contraction (approximately 5% MVC) and was defined as the lowest stimulus intensity able to induce MEP with greater than 100 μV peak-to-peak amplitude in FDI in at least 5 of 10 consecutive trials . The 1 mThe effect of muscle contraction strength on MEP and CSP induced by single-pulse TMS was investigated in 11 subjects . The subjects sustained the target EMG in isolation at 10%, 30%, 50%, and 70% EMG for 40 s. TMS intensity was 100% AMT, and 8 stimuli were applied at 0.2 Hz. A representative TMS trigger and rectified and smoothed EMG during sustained 10% EMG in experiment 1 are shown in Figure We investigated the effect of stimulus intensity on MEP and CSP in the same 11 subjects as in experiment 1. Right index finger abduction was performed in the same manner as in experiment 1. The muscle contraction level was 10% EMG, and TMS intensity was set at 100%, 110%, 120%, 130%, 140%, 150%, and 160% AMT with 0.2 Hz frequency. Muscle contraction was sustained for 40 s, and eight stimuli were applied at each TMS intensity level.The effects of SICI and ICF induced by paired-pulse TMS on MEP amplitude and CSP duration were investigated in 11 subjects . The stimulus intensities used for paired-pulse TMS were 80% AMT as a conditioning pulse and 1 mV TMS intensity as the test pulse. These conditioning and test intensities were the same for all muscle contraction levels, because CSP duration might be influenced by TMS intensity . ISI of MEP amplitudes were calculated from peak-to-peak amplitudes, except for the amplitude of the maximum and minimum MEP amplitude of 8 waves. CSP duration was defined as the time from TMS onset to the time of reappearance of EMG amplitude that was more than 3-fold the SD of the background EMG noise at rest, and the average values were calculated for all CSP durations. The decrease and increase ratios with SICI and ICF, respectively, [absolute (conditioned MEP − unconditioned MEP)/unconditioned MEP × 100) were calculated at each muscle contraction level.Statistical analysis was performed using PASW statistics 18 software . All data, including MEP amplitudes and CSP durations, were statistically analyzed by one-way repeated measures ANOVA with muscle contractions, stimulus intensities, and ISI as the within-subject factors. The sphericity of data was analyzed by Mauchly’s test, and Greenhouse–Geisser-corrected significance values were used when sphericity was lacking. Post hoc analysis was performed with Bonferroni’s methods for multiple comparisons to avoid type I errors. The correlations between MEP amplitude and the duration of CSP induced by single-pulse TMS and after SICI and ICF at each muscle contraction level were assessed by Pearson’s correlation analysis. Differences were considered significant at p < 0.05 for all analyses.CSP: Cortical silent period; TMS: Transcranial magnetic stimulation; MEP: Motor evoked potential; SICI: Short-interval intracortical inhibition; ICF: Intracortical facilitation; AMT: Active motor threshold; EMG: Electromyogram; SD: Standard deviation; SEM: Standard error of the mean; ISI: Interstimulus intervals; MVC: Maximum voluntary contraction; ANOVA: Analysis of variance; FDI: First dorsal interosseous.The authors declare no competing interests.SK conceived of the study, designed the experimental paradigm, performed the statistical analysis, and contributed to the discussion and preparation of the manuscript. HO conceived of the study, designed the experimental paradigm, performed the statistical analysis, and contributed to the discussion and preparation of the manuscript. KS conceived of the study, designed the experimental paradigm, performed the statistical analysis, and contributed to the discussion and preparation of the manuscript. HK conceived of the study, designed the experimental paradigm, and contributed to the discussion of the manuscript. MS performed the statistical analysis and contributed to the discussion and preparation of the manuscript. HT conceived of the study, designed the experimental paradigm, and contributed to the discussion of the manuscript. All authors read and approved the final manuscript.


The aim of the present study was to test the effects of PNU-282987 on spatial learning and memory and hippocampal neurogenesis in both intact and chronically stressed transgenic mice. Transgenic mice with susceptibility to Alzheimer's disease (AD) under immobilization stress and not-stressed animals receiving 0 and 1 mg/kg of PNU-282987 (PNU) were evaluated in a water maze task. The effects of PNU and stress on proliferation of new cells in the hippocampus of these animals were also assessed. The latency to escape the platform was significantly higher in transgenic stressed mice compared to those in the wild stressed group, as well as in transgenic animals without PNU compared to control wild group. On retention of the task, differences emerged on stressed wild animals, PNU wild group, and stressed wild mice receiving PNU. However, no significant differences were detected on new cell proliferation. The results of the present study did not show any impact of stress in acquisition of a spatial task both in wild and transgenic mice. No clear effects of PNU on acquisition of a spatial task in transgenic mice with susceptibility to AD were detected. Although PNU and stress effects were detected on retention of the task in wild animals, no changes were noted in transgenic mice. One of the most known hypotheses about the etiology of AD suggests that neurodegeneration begins with an abnormal processing of amyloid precursor protein (APP), resulting in production, aggregation, and deposition of the peptide Al death  . Among gctively  . These fβ peptide. These animals develop β-amyloid deposits in the brain at 6-7 months of age, showing a cognitive impairment at 12 months  [Nowadays, multiple strains of transgenic mice with mutations of the peptide amyloid precursor protein (APP) have been developed. They have shown to be very useful in studying this neurodegenerative disorder. Specifically, the transgenic strain B6C3-Tg  85 dB/J is a double transgenic model expressing a chimera of the amyloid precursor protein (Mo/HuAPP695swe) and a mutation of the human presenilin 1 (of 9-PS1). Both mutations are associated with early onset AD. The “humanized” transgene Mo/HuAPP695swe allows mice to secrete a human A months  , 5. Moreβ protein in a transgenic model of AD  [Among environmental factors that may contribute to AD, stress is one of the most important. Stress is currently an unavoidable condition in our daily experiences, activating physiological systems and disregulating the body homeostasis  , 8. Furtl of AD  . Anotherl of AD  .α7 nicotine acetylcholine receptor (nAChR) subtype seems to mediate the protective effects of nicotine  [β4α2 nAChRs decreases in AD, while that of α7 remains largely intact, being available for binding to new therapeutic agents  [α7 nAChRs do not seem to be related to the addictive effects of nicotine  [α7 nAChRs  [α7 nAChR agonists have been partial agonists of these receptors  [α7 nAChR agonists. Only a few studies on the behavioral effects of this drug have been published. Thus, Bitner and coworkers  [α7 nAChR-selective agonist, enhanced the cognitive performance of monkeys, rats, and mice in behavioral studies. In turn, the α7 nAChR subtype was specifically shown to be involved in human memory function  [α7 nAChR, having negligible interactions with other nAChR subtypes and being currently the most specific α7 nAChR agonist synthesized  [Cholinergic neurodegeneration is considered a significant mechanism of cognitive deterioration in elderly and AD patients  . Agents icotine  –16. On t agents  . Moreoveicotine  . Preclin nAChRs  , 20. Accceptors  . Little workers   found thunction  . Recent unction  . This efunction   or to anunction  . PNU-282hesized  . α7 subunit of the nAChR has been identified as a meaningful intermediary of nicotine's interaction with the stress axis and human disease  [α7 mRNA and decreased receptor binding at hippocampal formation, which suggests that nAChR α7 may be important for the adaptation to stress or hippocampus allostatic load  [As suggested by Craig and coworkers  , the neudisease  , among oic load  .One of the most interesting plastic processes taking place in the hippocampus is neurogenesis. The concept of neurogenesis was introduced more than 40 years ago  . Howeverα7 agonist as well as the effects of those variables on proliferation of new cells in hippocampus were also investigated.The present study was aimed at evaluating the possible effects of stress in precipitating the onset of deficits in spatial learning and memory in animals with susceptibility to AD. The possible therapeutic roles of PNU  n = 9).Wild SAL: 0.9% saline, not subjected to restraint stress, wild animals ( n = 9).Wild SAL-STR: 0.9% saline, subjected to restraint stress, wild animals ( n = 10).Wild PNU: PNU, not subjected to restraint stress, wild animals ( n = 11).Wild PNU-STR: PNU, subjected to restraint stress, wild animals ( n = 10).Tg SAL: 0.9% saline, not subjected to restraint stress, transgenic animals ( n = 9).Tg SAL-STR: 0.9% saline, subjected to restraint stress, transgenic animals ( n = 9).Tg PNU: PNU, not subjected to restraint stress, transgenic animals ( n = 10). Tg PNU-STR: PNU, subjected to restraint stress, transgenic animals  85dB/J transgenic male mice  aged 2 months were used in this study. Animals were quarantined for 10 days after shipping and housed in plastic cages in an animal room, which was maintained at a temperature of 22 ± 2°C, a relative humidity of 50 ± 10%, and on a 12 h light/dark automatic light cycle (lights on: 08.00 AM–08.00 PM). All animals were allowed free access to food  and tap water. Animals were randomly divided into the following 8 treatment groups.The experimental design of the present study was approved by the Animal Care and Use Committee of the Rovira i Virgili University , following the “Principles of laboratory animal care.” They were carried out in accordance with the European Community Council Directive (86/609/EEC).α7 nAChR agonist PNU was purchased from Sigma-Aldrich (Barcelona). The drug was intraperitoneally administered at 1 mg/kg just before the behavioral tests. The results of a previous study suggested that only at this dose PNU-282987 significantly improved retention in the water maze [ The ter maze . PNU was Animals were restrained three times per day (30 min each time) during 30 days. A fixed time gap of 3 h was established between the restraint sessions. The restraint procedure consisted in placing the mice in metacrylate cylindrical holders from Letica Scientific Instruments . Animals were maintained in a prone position. In previous studies, this procedure clearly caused stress in pregnant rodents –47.  To evaluate spatial learning and memory, after a month of restraining animals, were subjected to the Morris water maze test at 3 months of age.  The water maze consisted of a circular tank , divided into four quadrants. An escape platform (diameter 10 cm) was located 1 cm below the surface of the water in the target quadrant. Animals performed 4 trials per day for 5 consecutive days. During each trial, mice were allowed 60 s to find the hidden platform and to remain on it for 30 s. If the animal failed to find the platform within this period, it was placed on it by the experimenter. The order of the three starting positions was randomized throughout the day for each mouse. To avoid proximal cues and prevent egocentric learning, an internal mobile wall was added to the maze, being the wall randomly moved between trials. This seems to increase Morris water maze sensitivity . At the μm thick, were cut with a cryostat and collected according to a fractionator principle [To evaluate hippocampus cell proliferation, two days after behavioral testing, 3 or 4 mice from each group were intraperitoneally injected with 5-bromo-2-deoxyuridine (BrdU)  at 100 mg/kg/day during two consecutive days . One dayrinciple . Samples2O2-TBS. Sections were then treated for DNA denaturation incubating in 2 M HCl at 37°C and rinsed in 0.1 M sodium borate buffer. Sections were blocked in TBS-Plus containing 3% normal goat serum and 1% Triton-X in TBS for 30 min. Antibody against BrdU  was diluted 1 : 500 in blocking buffer and incubated overnight at 4°C. Following this incubation, tissue sections were washed with TBS-Plus and incubated with secondary antibody  for 2 h. After additional washes, the secondary antibody was detected using the avidin-biotin complex reaction (ABC Elite Kit). Diaminobenzidine  was used as chromogen. Sections were thoroughly washed, mounted, and cover-slipped.One out of six sections was taken for biotinylated-BrdU immunostaining. Free-floating coronal sections of brain were rinsed in TBS and inactivated for endogen phosphatase activity in 0.6% Hhttp://www.mbl.org/. The total numbers of BrdU positive cells in the granular cell layer and hilus of the bilateral entire hippocampus were exhaustively counted in serial coronal brain sections. Each section was 240 μm apart from each other. The set of selected sections represents one-sixth of the whole hippocampus, being representative of the total hippocampus. Positive cells were counted by means of an optic microscopy  through a 100× objective. Cells in the uppermost focal plane were discarded to avoid counting twice cells cut in two parts [Data for proliferation were obtained by using methods of unbiased stereology. The hippocampus was identified by anatomical criteria following the Mouse Brain Library Atlas  availablwo parts . As usuawo parts .F and Post hoc Dunnett's T3 test were used when appropriated. One-way ANOVA for repeated measures was used to analyze spatial learning during the acquisition of the water maze. To analyze the differences between groups, a Bonferroni-adjusted pairwise comparison was used when necessary. Student's t-test was used for searching differences between transgenic mice and their respective wild type control groups. Nonparametrical analysis for cell proliferation was used. Statistical significance was set at P < 0.05.Behavioral data were analyzed by means of a three-way ANOVA (Genotype × Stress × Drug). Post hoc Tukey test was used to analyze the differences between groups. Analyses for variance homogeneity were performed by means of Levene's test. A Welch's F = 6.162; P < 0.001), Day  = 70.509; P < 0.001), Day × Group  = 2.776; P < 0.001), and Day × Genotype  = 15.383; P < 0.001) during acquisition of the water maze. Post hoc Tukey test showed that the latency to escape the platform was significantly higher in stressed and nonstressed transgenic mice (P = 0.038 and P = 0.015), in comparison to the corresponding wild mice groups (P < 0.001), 4 (P = 0.013), and 5 (P = 0.044) on stressed animals but only on days 3 (P < 0.001) and 4 (P = 0.013) in control animals  = 4.679; P = 0.020). A significant difference between wild and transgenic mice receiving PNU  was observed on the time spent in the target quadrant , PNU wild group , and stressed wild mice given PNU   .No significant differences were detected on new cell proliferation , while nβ protein in a transgenic model of AD [α7 cholinergic agonist, was evaluated. The effects of stress and PNU on the proliferation of new cells in hippocampus of animals with susceptibility to AD were also assessed. Since cholinergic neurodegeneration has been related to cognitive deterioration in AD , agents el of AD . The aimThe results indicated that although all animals learned the water maze, differences in the learning curve were noted. Although transgenic mice acquired more slowly the task, they reached similar levels as wild mice. Significant differences only emerged at the end of the learning period between wild and transgenic stressed animals, indicating a differential effect of stress depending on the genetic characteristics of the mice. It is important to consider that the current results showed a deficit in learning process in transgenic control mice compared to wild animals given 0.9% saline only . Stress With regard to spatial memory, no significant differences were observed between any experimental condition, neither in wild mice, nor in transgenic mice. Notwithstanding, a significant difference was detected between wild and transgenic mice receiving PNU. In order to better understand this difference, another analysis was performed to compare the time spent in the target quadrant with the mean time spent in the other three quadrants , 56. StrNo significant differences in new cell proliferation were found, which indicated no effects of stress on this neurogenesis process. In the scientific literature, multiple protocols to induce stress to mice may be found . Howeverα7 nAChR antagonist. In turn, PNU and stress effects were detected on retention of the task in wild type animals, but no changes were detected in transgenic mice. To better understand interactions between environmental and genetic factors in AD, further studies are clearly necessary. According to the results of the present study, a longer PNU administration and/or older animals could be used for determining whether stress and PNU effects are modified by age.In summary, the current data suggest possible effects of PNU on acquisition of a spatial task in transgenic mice, which should be more accurately evaluated. In this sense, it should be interesting to test the effects of an


Peritassa campestris (Celastraceae) was achieved from seed cotyledons cultured in semisolid Woody Plant Medium (WPM) supplemented with 2% sucrose, 0.01% PVP, and 4.0 mg L−1 IBA. Culture period on accumulation of biomass and quinone-methide triterpene maytenin in adventitious root were investigated. The accumulation of maytenin in these roots was compared with its accumulation in the roots of seedlings grown in a greenhouse (one year old). A rapid detection and identification of maytenin by direct injection into an atmospheric-pressure chemical ionization ion trap tandem mass spectrometer (APCI-IT-MS/MS) were performed without prior chromatographic separation. In vitro, the greatest accumulation of biomass occurred within 60 days of culture. The highest level of maytenin—972.11 μg·g−1 dry weight—was detected at seven days of cultivation; this value was 5.55-fold higher than that found in the roots of seedlings grown in a greenhouse.Establishment of adventitious root cultures of  Quinone-methide triterpenes (QMTs) are secondary metabolites that occur only in plants of the Celastraceae family. Therefore, QMTs are considered chemotaxonomic indicators of this family , 2. ThesDespite the pharmacological potential of maytenin, no reports of their chemical synthesis exist. This is probably because they are structurally complex and contain several stereogenic centers. Hence, extraction from plants may be a feasible strategy to obtain maytenin.Peritassa campestris  is a fruiting shrub native of the Brazilian savanna biome, known as Cerrado; this plant is a source of many QMTs patterns –24 was cμg DW mL−1) and filtered through a 0.22 μm membrane of a nylon filter. The solution was directly introduced into the APCI source by flow injection at 5 μL min−1, using a syringe pump. The MS and MS/MS analyses were accomplished on a Thermo Scientific LCQ Fleet ion trap spectrometer equipped with an APCI interface operating in the positive ion mode. The stainless steel capillary temperature was set to 200°C; the pressure of the nebulizing gas (nitrogen) was 88 psi.The root extracts were dissolved in methanol (1.0 −1). Next, a 1 mL aliquot of the resulting solution was submitted to solid-phase extraction (SPE) SPE cartridges (Sampliq Agilent) (3 mL) containing 500 mg of C18 silica gel (particle size of 40 μm) preconditioned with methanol and methanol/water (95 : 05 v/v). Cartridges were eluted with 3 mL of methanol/water (95 : 05 v/v). The interfering compounds were retained by the C18 silica gel, whilst the eluate was transferred to a 5 mL volumetric flask, adjusted to the mark with the same 95% methanol. The resulting solution was filtered through a 0.22 μm membrane of a nylon filter, and an aliquot (20 μL) was assayed by HPLC.The root extracts were dissolved in methanol/water  . The mobile phase was methanol/water/formic acid ; elution was carried out in the isocratic mode for 20 min, at a flow rate of 1 mL min−1. The injection volume was 20 μL for the samples and the chemical standard; detection was conducted at a wavelength of 420 nm. Maytenin (HPLC purity > 98%), isolated and identified by NMR and MS experiments in our laboratory from the root barks of P. campestris, was employed as chemical standard. Measurements were integrated by comparison with an external standard calibration curve. Limits of detection and quantification were, respectively, 41,00 × 10−3 and 124,0 × 10−3 μg·mL−1.The content of maytenin in the root extracts was determined using the method described by Corsino at al.  with a fin vitro plant culture techniques—it is simple, provides homogenous and fast growing material, and is easy to scale up. However, the biosynthetic machinery necessary to generate the various target metabolites may not be available or may afford only small amounts of the desired compounds. In such an event, a plant organ culture might be a preferable system [Plant cell suspension cultures are the most popular strategy among the e system .P. campestris after four weeks of inoculation in WPM supplemented with 2% (w/v) sucrose, 0.01% (w/v) PVP, and 4.0 mg·L−1 IBA  peaks at seven days. A similar growth pattern was observed for a culture of Maytenus ilicifolia (Celastraceae) callus—production of this same metabolite peaked in the log phase  [μg·g−1 DW). Buffa-Filho et al. [M. ilicifolia callus produce lower amounts of this QMT in the same growth phase. These results were expected since plant cells usually produce primary metabolites at the expense of secondary metabolites during the linear growth phase.The increased production of maytenin during the first seven days of culture does not correspond to accumulation of adventitious root biomass. The content of maytenin (972.11 ·g−1 DW) . During o et al.  also verμg·g−1 DW); in the subsequent 30 days, it decreases (620.83 μg·g−1 DW), whereas the adventitious root remains in the stationary phase. After 90 days of cultivation, the adventitious roots begin to decline, and the concentration of maytenin increases slightly.Between 45 and 60 days of cultivation, the concentration of maytenin increases  and the adventitious roots biosynthesize maytenin, however in different amounts. After seven days of in vitro culture (peak of production), the adventitious roots produce 5.55 times more maytenin than the roots of seedlings .Analyses reveal that both, the roots from seedlings ofin vivo plants is not a rule. The concentration of any metabolite may be higher or lower, depending on factors such as light, culture medium, and growth regulators, among others [P. campestris, the greater accumulation of QMTs by the in vitro adventitious root may be associated with the presence of IBA. Accordingly, [Higher production of secondary metabolites in tissue cultures as compared with g others –30. Concrdingly,  auxins a in vitro lab-scale plant cultures, the most suitable method should be fast and highly sensitive, given the limited quantities of biomass. MS is a method that meets all these requirements, even in the absence of reference standards [A variety of screening methods exist to detect and identify known and unknown metabolites in plant materials . Howevertandards .P. campestris  sucrose, 0.01% (w/v) PVP, and 4.0 mg·L−1 IBA under dark conditions. The productivity of maytenin in the in vitro roots is higher comparing with the in vivo production by the roots of seedlings. These results indicate that the adventitious roots cultures of P. campestris are a promising source of maytenin and a base for scaling-up adaptations in view of its future production in bioreactors.An efficient protocol to grow adventitious roots of P. campestris are a fast, simple, and efficient strategy to screen the QMT maytenin.APCI-IT-MS/MS analyses using direct flow injection of the root extracts of MS, 1H, and 13C NMR (1D and 2D) spectra of maytenin.Click here for additional data file.


Previous research has provided considerable support for idea that increased parental support and control are strong determinants of lower prevalence levels of adolescent risk behavior. Much less is known on the association between specific parenting practices, such as concrete rules with respect to smoking and drinking and adolescent risk behavior. The present paper examined whether such concrete parental rules (1) have an effect on the targeted behaviors and (2) predict other, frequently co-occurring, risk behaviors . These hypotheses were tested in a nationally representative sample of 12- to 16-year-old adolescents in the Netherlands. We found that both types of rules were associated with a lower prevalence of the targeted behaviors . In addition, independent of adolescent smoking and drinking behaviors, parental rules on smoking predicted a lower prevalence of cannabis use and early sexual intercourse, and parental rules on alcohol use also predicted a lower prevalence of early sexual intercourse. This study showed that concrete parental rule setting is more strongly related to lower levels of risk behaviors in adolescents compared to the more general parenting practices . Additionally, the effects of such rules do not only apply to the targeted behavior but extend to related behaviors as well. These findings are relevant to the public health domain and suggest that a single intervention program that addresses a limited number of concrete parenting practices, in combination with traditional support and control practices, may be effective in reducing risk behaviors in adolescence. In many Western countries, adolescent risk behaviors are among the top priorities in the public health domain. Specifically, numerous prevention and intervention programs have been developed to reduce young people’s engagement in smoking tobacco, drinking alcohol, using cannabis, and early sexual intercourse. In recent years, researchers have observed two trends in the development of such programs. First, public health programs aiming to reduce adolescent risk behaviors have increasingly focused on the role of parents  studies that have focused on general parenting practices, most notably parental support and control Baumrind  and (2) With respect to specific parenting practices, most research has focused on practices that target adolescent smoking and drinking. Among specific parenting practices aimed at reducing adolescent alcohol use, the strongest predictor is concrete parental rules on adolescent drinking behavior  also have an effect on other, related risk behaviors .In the present study, we examined these research questions based on a large, nationally representative sample of Dutch adolescents in secondary education. Consistent with existing literature, we hypothesized that parental support would be negatively associated with adolescent smoking, drinking, cannabis use, and early sexual debut. Furthermore, we expected parental control to be negatively associated with all four risk behaviors; however, this association would be mediated by concrete parental rules on smoking and drinking  survey. The HBSC study is a World Health Organization collaborative cross-national study on the health, health-related behaviors, and the social context of young people’s health. Consistent with the international study protocol  in the first 4 years of secondary education. 51 % of the respondents were boys, and 15 % had an ethnic minority background. With respect to educational level, 46 % of the respondents were classified as having a high educational level .The final sample included 5,642 students, who were representative of Dutch youth aged 12–16 years  were recoded into ‘no daily smoking’ and ‘daily smoking.’ As daily smoking is a crucial aspect of nicotine dependence Jarvis , daily sWith respect to alcohol use, adolescents were asked: ‘How often have you, in the previous month, drunk five or more alcoholic drinks on one occasion (for example at a party or a night out)?’ Original answer categories (ranging from ‘never’ to ‘nine times or more’) were recoded into ‘never’ and ‘at least once.’ Regular binge drinking is considered an indicator of excessive alcohol use  were recoded into ‘never’ and ‘at least once.’ Cannabis use is rare among adolescents aged 12–16; if adolescents at this age already have experience with cannabis use, this is generally interpreted as an (extreme) risk behavior. Early cannabis use has been found to affect neurocognitive functioning  as indicators of model fit. The CFI and TLI are related to the total variance accounted for in the model; values greater than 0.90 are accepted and values greater than .95 are desired , Tucker-Lewis Index (TLI), and the Root Mean Square Error of Approximation (ed Kline . The RMSed Kline . For an χ²(9) = 728.59, p = .00, CFI = .96, TLI = .94, RMSEA = .12.This construct was based on six indicators: (1) My parents show me that they admire me; (2) In my parents’ eyes, I do everything wrong; (3) My parents show me that they love me; (4) My parents often make me look ridiculous; (5) My parents support me in my activities; and (6) My parents treat me aggressively. Answer categories ranged from ‘definitely true’ to ‘definitely not true’  = 42.45, p = .00, CFI = .99, TLI = .97, RMSEA = .09.The construct parental control was based on three indicators: (1) Before you leave the house, do your parents want to know with whom or where you are going?; (2) Do you need your parents’ permission to go out at night?; and (3) If you go out at night, do your parents want to know afterward with whom or where you were? (adapted from Kerr and Stattin χ²(2) = 330.37, p = .00, CFI = 1.00, TLI = .98, RMSEA = .17.The construct alcohol-specific rules was based on four indicators: (1) I am allowed to drink one glass of alcohol when my father or mother is home, (2) I am allowed to drink several glasses of alcohol when my father or mother is home, (3) I am allowed to drink alcohol at a party with friends, and (4) I am allowed to drink alcohol on the weekends. Answer categories ranged from ‘definitely not true’ to ‘definitely true’  = 274.52, p = .00, CFI = 1.00, TLI = .99, RMSEA = .22.As previous research mainly focused on house rules with respect to smoking, new indicators for smoking-specific rules were developed. These indicators included: (1) I am allowed to try out smoking a cigarette, (2) I am allowed to smoke now and then, (3) I am allowed to smoke regularly. Answer categories ranged from ‘definitely not true’ to ‘definitely true.’ A confirmatory factor analysis revealed acceptable fit statistics: All analyses controlled for gender (boy vs. girl), age (ranging from 12 to 16), and educational level (low vs. high). Educational level, which is a strong predictor of adolescents’ future socioeconomic status ROA , was incFirst, we provide descriptive statistics to identify the prevalence of adolescent risk behaviors and the amount of parental support, control, and concrete rules on adolescent smoking and drinking behaviors for male and female adolescents, youth from the different age groups, and students with high and low educational levels.Second, we designed a structural equation model in Mplus version 6.11   boys and girls, (2) adolescents from different age groups (early vs. mid adolescents), and (3) adolescents with a low versus high educational level. For each moderation analysis, the model fit of two models was compared: (1) a model in which all paths were freely estimated for the two groups  and (2) a model in which the paths from the parenting practices to the risk behaviors were constrained to be equal across groups. The model fit comparison was based on two criteria, which both needed to be met in order to conclude that there was a significant difference between the models. These criteria were (1) the chi square difference test, a traditional test that indicates whether the fit of two models differs significantly but which is sensitive to sample size and (2) Chen’s criteria, which are not sensitive to sample size  Chen . If the N of our sample was large, we used significance criteria of α = .001 and α = .01.Data were weighted for educational level, grade, gender, and urbanicity with poststratification weights. All analyses were corrected for cluster effects of pupils within the same school (primary sampling unit). The range from missing values per variable ranged from 0 % to 3 % . A robust weighted least squares estimator (WLSMV) was used in combination with full information maximum likelihood estimation to deal with missing data  = 875.42, p = .000, CFI = .99, TLI = .99, RMSEA = .03. The model estimates of the final model are illustrated in Fig. Our model, which aimed to predict adolescent risk behaviors by the four parenting practices, had a good fit: χ²84 = 875.β = -.20, p < .001), lifetime cannabis use , and early sexual intercourse . In addition, via the mediator parental rules on alcohol use, parental control had an indirect effect on adolescent binge drinking  and early sexual intercourse .Parental support was negatively associated with all four risk behaviors. There was no direct association between parental control and risk behavior; however, via the mediator parental rules on smoking, parental control had an indirect effect on adolescent daily smoking  and β = -.60 (smoking rules and adolescent smoking behavior). Interestingly, the associations with other behaviors were also quite strong and ranged from β = -.13  to β = -.35 (smoking rules and adolescent cannabis use).The direct associations between parental rules on smoking and drinking were especially high with the targeted behaviors: r = .31  to r = .45 (smoking and cannabis use) (ps < .001).Furthermore, parental support and parental control were strongly correlated, as were parental rules on adolescent smoking and drinking. The four risk behaviors were all correlated and ranged from In total, this model explained 47.3 % of the variance in binge drinking, 61.5 % of the variance in daily tobacco smoking, 44.0 % of the variance in lifetime cannabis use, and 39.4 % of the variance in early sexual intercourse.χ² (16) = 22.09, p = .14) and Chen’s criteria  showed that the fit of the freely estimated model (χ²(400) = 1148.97, p < .001, CFI = .99, TLI = .99, RMSEA = .03) was not significantly better than the fit of the constrained model (χ²(416) = 1106.70, p < .001, CFI = .99, TLI = .99, RMSEA = .02), which indicates that the associations between parenting practices and adolescent risk behaviors were similar for early and mid adolescents.To test whether the results of our model applied similarly to different subgroups of youth, three moderation analyses were conducted. For each analysis, the model fit of the model in which all regression paths were constrained across the groups, was compared with the freely estimated model. In the analysis that compared early versus mid adolescents, both the chi square difference test (Δχ² (16) = 21.37, p = .16) indicated that the freely estimated model had a better fit; however, Chen’s criteria  did not support this finding. The fit of both models was good: χ²(400) = 1194.10, p < .001, CFI = .99, TLI = .99, RMSEA = .03 for the freely estimated model and χ²(416) = 1189.12, p < .001, CFI = .99, TLI = .99, RMSEA = .03 for the constrained model. Based on the fact that the chi square test is sensitive to sample size and, on the observation that the more robust fit indices were hardly different (Chen’s criteria), we concluded that the constrained model was preferred, which indicates that the associations in our model did not differ for boys and girls.In the gender analysis, the chi square difference test (Δχ²(416) = 983.83, p < .001, CFI = 1.00, TLI = .99, RMSEA = .02) and freely estimated model (χ²(400) = 1023.57, p < .001, CFI = .99, TLI = .99, RMSEA = .03) did not significantly differ based on the chi square difference test (Δχ² (16) = 18.64, p = .29) or Chen’s criteria , which indicates that the associations were also similar for students with a low and high educational level. In conclusion, the associations in our model were similar in strength for all subgroups of youth.Finally, in the analysis that compared adolescents with a low and high educational level, the model fit of the constrained  were associated negatively with adolescent smoking, drinking, cannabis use, and early sexual activity. Second, concrete parental rules on smoking were associated with a lower likelihood of adolescent smoking and concrete parental rules on alcohol use were associated with a lower likelihood of adolescent drinking. These effects were stronger than the effects of parental support and control. Third, parental rules also had an effect on other risk behaviors: rules on alcohol use predicted a lower likelihood of early sexual intercourse and rules on smoking predicted a lower likelihood of adolescent cannabis use and early sexual intercourse. Finally, the abovementioned associations were equally strong for early and mid adolescents, boys and girls, and students with low and high educational levels.The first finding, that adolescent smoking, drinking, cannabis use, and sexual activity were negatively influenced by parental support and control, confirmed our hypothesis and is consistent with previous research . A final explanation is that parents who set rules with respect to adolescents’ smoking and drinking behaviors, generally tend to monitor their children more so compared to parents who do not set such rules. This would explain why, for example, parental rules on smoking and adolescent delay in sexual debut are associated, even though they are not very close conceptually.The finding that the parenting practices under study were related to a reduction in adolescent risk behaviors similarly for early and mid adolescents, boys and girls, and youth with different educational levels, was somewhat surprising. Specifically, the findings show that, in subgroups of youth who experience less parental support, control, and concrete rules , the effects of these parenting practices are similar. Similarly, for subgroups of youth who may spend less time with their parents and more time with peers  in the Netherlands. Second, our model explained a relatively large part of the variance in adolescent risk behavior, which indicates that our predictors  contribute substantially to explaining adolescent engagement in risk behaviors. Finally, this study does not only have implications for academia, but also for the public health domain. It is our hope that the current results can guide prevention workers toward the development of effective and efficient prevention programs.Our study also has a few limitations. First, it was based on cross-sectional data; therefore, we cannot make any causal inferences. Based on our findings, it is not clear whether parenting practices influence adolescent behaviors, whether adolescent behaviors influence parenting practices, or whether there is a third variable that influences both parenting practices and adolescent risk behaviors. Factors that may impact both parental support, control, and rule setting and adolescents’ propensity for risk behavior include genetics  may be effective in simultaneously reducing adolescent substance use and early sexual intercourse among 12 to 16-year-old youth. Such broad programs would be more cost efficient and probably have higher rates of parental compliance compared to the implementation of separate programs that target a single risk behavior.It is important to note that an intervention study is necessary to actually conclude that such a program would be beneficial. Currently unknown factors may play a role in causing both parents to adopt certain parenting practices and adolescents to engage less in risk behaviors. Additionally, based on the current data, we do not know how parents who do not engage in certain practices would react if they were encouraged to adopt these practices. If an intervention study succeeds in demonstrating that parenting practices can be modified and that this has a desired effect on adolescents, only then could such a program be deemed as evidence-based and be implemented. Therefore, the current study should be perceived as a modest step toward exploring the possibility of designing broader intervention programs that reduce adolescent smoking, drinking, drug use, and early sexual intercourse.


Huang-Lian-Jie-Du-Tang (HLJDT), a classical Chinese prescription, has been clinically employed to treat cerebral ischemia for thousands of years. Geniposide is the major active ingredient in HLJDT. The aim is to investigate the comparative evaluations on pharmacokinetics of geniposide in MCAO rats in pure geniposide, geniposide : berberine, and geniposide : berberine : baicalin. Obviously, the proportions of geniposide : berberine, geniposide : baicalin, and geniposide : berberine : baicalin were determined according to HLJDT. In our study, the cerebral ischemia model was reproduced by suture method in rats. The MCAO rats were randomly assigned to four therapy groups and orally administered with different prescription proportions of pure geniposide, geniposide : berberine, geniposide : baicalin, and geniposide : berberine : baicalin, respectively. The concentrations of geniposide in rat serum were determined using HPLC, and main pharmacokinetic parameters were investigated. The results indicated that the pharmacokinetics of geniposide in rat serum was nonlinear and there were significant differences between different groups. Berberine might hardly affect the absorption of geniposide, and baicalin could increase the absorption ability of geniposide. Meanwhile, berberine could decrease the absorption increase of baicalin on geniposide. Rhizome coptidis (9 g), Radix scutellariae (6 g), Cortex phellodendri (6 g), and Fructus gardenia (9 g). Modern pharmacological studies have shown that HLJDT decoction can prevent cerebral ischemia and antioxidation . In . In Rhizcoptidis  g, RadixThere are two other major constitutes, alkaloids (berberine) and flavonoids , existing in HLJDT . So it iGeniposide and paeoniflorin were purchased from National Institute for the Control of Pharmaceutical and Biological Products . The extracts of geniposide, baicalin, and berberine were purchased from Zelang Biotechnology Company . Methanol used for HPLC was chromatographic grade . Deionized water was prepared in a Mill-Q academic water purification system . Glacial acetic acid was analytical grade, which was provided by Jiuyi Chemical Reagent Company . Heparin sodium was purchased from Sigma. Acetonitrile, HPLC grade was purchased from Merck.3PO4 (B) with isocratic eluting  at a flow rate of 1.0 mL/min. The detector operated at 238 nm. The injection volume was 20 μL and the column temperature was 30°C.The High Performance Liquid Chromatography (HPLC) system consisted of a Waters 515 pump, DAD detector ; N2000 LC chromatography workstation . The mobile phase was Acetonitrile (A) and 0.1% HThe experiment mainly studied the influence of baicalin and berberine on the pharmacokinetic behavior of geniposide. According to the preliminary experiments, the average contents of geniposide, baicalin, and berberine were 22.82, 40.02, and 25.78 mg/g in HLJDT. Pure geniposide, geniposide : beberine, geniposide : baicalin, and geniposide : beberine : baicalin were weighed accurately on the basis of proportion in HLJDT, and then dissolved in 0.50% carboxymethyl cellulose sodium (CMC-Na) aqueous solution before use.Male Sprague-Dawley (SD) rats were purchased from the slaccas experiment animal company . Animal welfare and experimental procedures were strictly in accordance with the Guide for the Care and Use of Laboratory Animals  and the related ethics regulations of Nanjing University of Chinese Medicine.Experiments were conducted in 290 ± 10 g SD rats under chloral hydrate anesthesia and fixed the rats on the operating table. A ventral midline incision was made in the neck. The omohyoid muscle was separated longitudinally and retracted laterally and isolated. A loose ligature was then placed around the external cartid artery. The external artery was permanently ligated rostrally. The vessel was ransected between the ligatures, and the remaining stump was reflected caudally. A piece of monofilament suture material was inserted into the lumen of the stump and advanced into the internal carotid artery. When completely advanced, the tip of the monofilament should block the blood flowing into the middle cerebral artery. The silk ligature was tied around the stump to secure the piece of monofilament. The skin irrcision was closed using wound clips. Four hours after surgery, as a successful replication of the model of MCAO, the body temperature will rise over 0.8°C, and rats showed visible symptoms of neurological deficit which are characterized by severe left-sided hemiparesis and right Horner's syndrome , 14. It The MCAO rats were divided into four groups randomly, pure geniposide subgroup, geniposide : berberine subgroup, geniposide : baicalin subgroup, and geniposide : baicalin : berberine subgroup. Pure geniposide was dissolved in 0.5% CMC-Na aqueous solution and was oral to the pure geniposide subgroup rats (containing 142.28 mg geniposide/kg according to body weight). The geniposide : berberine subgroup, geniposide : baicalin subgroup, and geniposide : baicalin : berberine subgroup also received gavages of geniposide dissolved in 0.5% CMC-Na aqueous solution at the dosage of containing 142.28 mg geniposide/kg according to body weight. Blood samples (0.5 mL) were collected from the abdominal vein before dosing (to serve as a control) and at 0.083, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 12.0, and 24.0 h after drug administration, then immediately transferred into EP tube and centrifuged for 5 min at 5000 rpm in 4°C to separate serum. The serum was stored at −70°C after separation until assayed as described below.μL) were acidified with approximately 100 μL of 0.01 mol/L acetic acid added to each of them. Then, each portion was vortexed with 800 μL of methanol for 1 min and centrifuged at 5000 rpm for 10 min. The organic layer was transferred into an empty tube and was dried at 40°C under a nitrogen stream. The residue was dissolved in 100 μL methanol. After centrifugation at 800 rpm for 10 min, fifty microliters supernatant was analyzed with HPLC.Serum samples , medium (0.58 μg/mL), and high (2.32 μg/mL) concentrations for geniposide. It was calculated by comparing the mean peak area (n = 5 at each concentration) of the extracted quality control sample with that of the unextracted standard solution containing the equivalent amount of analyses.Recovery of the liquid-liquid extraction procedure was evaluated at low  and the precision was evaluated by the relative standard deviation (RSD).Quality control (QC) samples were used for the study of intraday and interday accuracy and precision; extraction recovery and stability were prepared in the same way as above and were prepared from blank serum at concentrations of 0.15, 0.58, and 2.32 The stability of geniposide was evaluated under mimicking conditions likely to be encountered during sample storage and the analytical process by analyzing five replicates of QC samples for the analysis. The freeze-thaw stability was determined after one freeze and thaw cycle. The QC samples were stored at −20°C for 24 h and thawed unassisted at room temperature.P value of less than 0.05 was considered statistically significant.The different serum concentrations of geniposide were expressed as means ± standard deviation (SD) and the mean concentration-time curve was plotted. All data were analyzed by software kinetica version 4.4  to obtain the relative pharmacokinetic parameters. Statistical analysis was performed using the SPSS Version 13.0 . Differences in numerical variables among groups were analyzed with one-way analysis of variance (ANOVA) LSD-t and SNK-q. A The selectivity of the method was evaluated by analyzing blank serum samples prior to administration. The chromatograms were free of interfering peak at the retention time of geniposide (13.3988 min); the retention time paeoniflorin (8.392 min) of μg/mL. The lower limit of quantification (LLOQ) for geniposide was 0.15 μg/mL.Each calibration curve was constructed with six different concentrations by plotting the peak areas ratios of geniposide versus the concentration of geniposide using linear regression. Good linearity was obtained from 0.15 to 9.26 The intra- and interday precision and accuracy were determined by replicate analyses of QC samples continuously on the same day (intraday) for 5 days (inter-day), respectively. The intra-and interday precision and accuracy were shown in μg/mL, respectively., 0.15, 0.58, and 2.32.The mean (±SD) recovery for geniposide from rat serum was 85.34 ± 2.04%, 81.92 ± 2.23%, 78.75 ± 3.69% at 2.32, 0.58, 0.15 μg/mL, respectively. Geniposide showed good stability in one week after storage at −20°C for 7 days, the concentration of geniposide in serum would deviate to less than ±15% from those in freshly spiked serum.The stability of geniposide was determined by analyzing QC samples at three concentrations exposed to encounter during sample storage. QC samples were frozen and stored at −20°C for a week. Freeze-thaw stability was determined after one cycle of freezing and thawing, the corresponding relative errors were less than ±10% for samples at the concentrations of 4.36, 3.45, and 8.79 t). These results indicated that baicalin increased the absorption of geniposide. Pharmacokinetic comparison of geniposide on MCAO rats after administration of geniposide and geniposide : berberine illustrated that the existence of berberine might hardly affect the pharmacokinetic behavior of geniposide.HLJDT contains effective parts of three categories: alkaloids, flavonoids, and iridoid glycosides, three active ingredients: berberine, baicalin, and geniposide, and so forth . The dev∞) and lower than that of geniposide : baicalin : berberine  and geniposide : baicalin . The data demonstrated that coadministration of geniposide with baicalin, the bioavailability of geniposide, increased significantly in MCAO rats body. It might be because baicalin inhibited the hydrolysis of geniposide. Berberine showed no significant influence on behaviors in the pharmacokinetics of geniposide in MCAO rats, but it decreased the bioavailability of coadministration of geniposide with baicalin. It might be because berberine partly neutralized baicalin and the hydrolysis on geniposide was enhanced.It investigated pharmacokinetic differences of coadministration geniposide with baicalin and/or berberine changing by time in cerebral ischemia rats. Using kinetica version 4.4 and SPSS Version 13.0 to process serum concentration data and using the method of statistical moments we calculated pharmacokinetic parameters –25. As stmax) was shorter, apparent volume of distribution (V) and concentration to peak (Cmax) were higher, the dwell time (MRT) was longer, clearance rate (CL) was lower, and so on, indicating that the absorption effects of geniposide in MCAO rats were better than that of normal rats. Hou et al. [β-glucuronidase held that the hydrolyzed enzymes might be reduced in MCAO rats. However, in the future, more experiments such as tissue distribution and metabolic pathway of geniposide in the body should be taken out for the purpose of exploring its absorption and metabolism mechanism.At the same time, we studied the pharmacokinetics of geniposide with or without baicalin and/or berberine in normal rats. The results had some differences from that of the MCAO rats. Compared with normal rats, in MCAO rat, the time to peak (0−, and Cmax. In the meantime, coadministration of geniposide with berberine could not significantly affect the pharmacokinetic parameters. However, the pharmacokinetic parameters of geniposide : baicalin were significantly decreased by berberine.The animal study showed significant comparative pharmacokinetics of geniposide alone or in combination with baicalin and/or berberine in rat model of cerebral ischemia. Coadministration of geniposide with baicalin could significantly increase the AUC


Cross-lagged path analyses demonstrated that adolescents’ GAD symptoms and perceived father-adolescent attachment relationship quality bidirectionally negatively affected each other over time. For mothers, adolescents’ GAD symptoms negatively predicted perceived mother-adolescent attachment relationship quality over time. The within-wave correlated residuals between perceived attachment relationship quality with fathers and GAD symptoms were stronger for boys than for girls and stronger for the cohort of middle adolescents than for the cohort of early adolescents. This study demonstrates that both the parents’ and the adolescents’ gender as well as the adolescents’ age affects the relation between adolescents’ GAD symptoms and perceived parent-adolescent attachment relationship quality.This longitudinal study examined the direction of effects between adolescents’ generalized anxiety disorder (GAD) symptoms and perceived parent-adolescent attachment relationship quality, as well as the moderating role of gender and age. 1,313 Dutch adolescents (48.5% boys) from two age cohorts of early ( Generalized Anxiety Disorder (GAD) is the most common anxiety disorder in adolescence  whether lower levels of perceived attachment relationship quality predict higher levels of GAD symptoms, (2) whether higher levels of GAD symptoms predict lower levels of perceived attachment relationship quality, (3) whether there is a bidirectional effect over time. Since this study is the first that examines the direction of effects in the relation between GAD symptoms and perceived attachment relationship quality, no specific hypothesis of the direction of effects was made. Furthermore, we investigated whether the relation between GAD symptoms and perceived attachment relationship quality differs for adolescent-father dyads and adolescent-mother dyads. Next, we examined whether gender moderates the relation between GAD symptoms and parent-adolescent attachment relationship quality. We hypothesized that the relation will be stronger for girls than for boys. Finally, we examined whether age differences exist in the association between GAD symptoms and perceived attachment relationship quality. We hypothesized that during early adolescence GAD symptoms are more strongly related to perceived parent-adolescent attachment relationship quality than during middle adolescence.n = 923) and a middle to late adolescence cohort (n = 390). The mean age at the first wave was 12.42 years (SD = 0.59) of the early adolescence cohort, and was 16.68 years (SD = 0.80) of the middle adolescence cohort. Because both age groups were assessed during five measurement waves, a total age range from 12 to 16 and from 16 to 20 years was available. The majority of the participants were Dutch (82.8%). The rest (17.2%) identified themselves as part of a non-Western ethnic group. The large majority of the adolescents (82.9%) lived with both parents. Sample attrition was 1.2% across waves, and 7.14% of the values was missing across waves. To estimate the pattern of missing values, Little’s  = 649.54, p = 0.00, the χ2 ⁄ df ratio of 1.58 indicated a good fit between sample scores with and without imputation . The SCARED is a self-report questionnaire designed for children and adolescents that measures the occurrence of anxiety disorder symptoms. The GAD subscale consists of 9 items. Example items are: “I worry if others will like me”, and “I worry about the future”. Adolescents rated each statement on a 3-point scale: 0 , 1 (sometimes), and 2 (often). Items were averaged to compute a mean score, with higher mean scores indicating greater reports of GAD symptoms. The scale has demonstrated good reliability and construct validity  of GAD symptoms, stability paths of perceived attachment relationship quality, as well as correlations between within-wave residuals of GAD symptoms and perceived attachment relationship quality. In subsequent models cross-lagged paths were added to the baseline model to investigate whether this improved the overall fit of the total model. The second model was a unidirectional model from adolescents’ GAD symptoms to perceived attachment relationship quality, implying that GAD symptoms in adolescents affect perceived parent-adolescent attachment relationship quality and not vice versa. The third model was also a unidirectional model, but in this model paths were drawn from perceived attachment relationship quality to GAD symptoms, implying that adolescents’ perception of attachment relationship quality with parents affect adolescents’ GAD symptoms and not vice versa. The last model was a bidirectional model with mutual paths between GAD symptoms and perceived attachment relationship quality, based on the assumption that there are bidirectional effects between these constructs. For the sake of parsimony we tested whether it was possible to fix over the waves the within-wave correlated residuals and the cross-lagged paths between adolescents’ GAD symptoms and perceived parent-adolescent attachment relationship quality.After determining which model best represented the data of the total sample, we used two series of multigroup analyses to test the moderation effects of gender and age on the associations between GAD symptoms and perceived attachment relationship quality. We constrained within-wave correlated residuals and cross-lagged paths between adolescents’ GAD symptoms and perceived attachment relationship quality to be equal across groups . When fixation of different paths to be equal significantly impaired the model fit, there were significant differences between the two groups, and we released the constraints again.For evaluating the fit of the models, several goodness-of-fit indices were used: the Comparative Fit Index (CFI), with values above 0.90 representing a satisfactory fit and values above 0.95 demonstrating a good fit; the Root Mean Square Error of Approximation (RMSEA), with values up to 0.10 indicating an acceptable fit and values up to 0.05 being indicative of a good fit  = 0.54, p = 0.76, and from perceived attachment relationship quality to GAD symptoms, ΔχSB²  = −3.64, p = 1.00. Note that although unstandardized coefficients were constrained to be equal, the standardized coefficients can still slightly differ from each other. It was not possible to constrain the paths from GAD symptoms to perceived attachment relationship quality to be equal to the paths from perceived attachment relationship quality to GAD symptoms, ΔχSB²  = 34.84, p < 0.01. It was also not possible to fix the within-wave correlated residuals across waves, ΔχSB²  = 8.19, p < 0.05. Model fit for the final model for fathers was: χSB² (16) = 182.17, p = 0.80; CFI = 0.94; RMSEA = 0.09.For all adolescents, we tested whether adding the cross-lagged paths between GAD symptoms and perceived attachment relationship quality Model 2, 3, and 4) improved model fit compared to a baseline model with only stability paths and within-wave correlated residuals (Model 1). For fathers, Models 2, 3, and 4 significantly improved model fit compared to the baseline model (Model 1). So we continued with Model 4 that has stability paths, within-wave correlated residuals and cross-lagged paths from GAD symptoms to perceived attachment relationship quality, as well as cross-lagged paths from perceived attachment relationship quality to GAD symptoms  = 1.63, p = 0.44. It was also possible to fix the within-wave correlated residuals, ΔχSB²  = 4.02, p = 0.13. Model fit for the final model for mothers was: χSB² (19) = 177.67, p = 0.22; CFI = 0.94; RMSEA = 0.08.For mothers, both Models 2  and 4  improved fit compared to the baseline model (Model 1). As Model 3  did not improve fit compared to the baseline model, and Model 4 did not improve fit compared to Model 2, we chose Model 2 as the best fitting model  = 21.86, p < 0.05. The models with different paths from GAD symptoms to perceived attachment relationship quality, ΔχSB²  = 0.63, p = 0.43, and with different paths from perceived attachment relationship quality to GAD symptoms, ΔχSB²  = 0.36, p = 0.55, did not provide a better fit. Model fit for the final model for fathers was: χSB² (34) = 201.98, p = 0.63; CFI = 0.94; RMSEA = 0.09.For perceived attachment relationship quality with fathers, the chi-square difference tests showed that the model with different paths for the groups provided a better fit to the data than the model with equality constraints across groups for within-wave correlated residuals, ΔχSB²  = 0.85, p = 0.35, and for paths from GAD symptoms to perceived attachment relationship quality, ΔχSB²  = 0.03, p = 0.98. Model fit for the final model for mothers was: χSB² (41) = 198.97, p = 0.84; CFI = 0.94; RMSEA = 0.08.For adolescents’ perceived attachment relationship quality with mothers, the model with different paths for the groups did not provide a better fit for the data than the model with equality constraints across groups for within-wave correlated residuals, ΔThus, there were only gender differences in the within-wave correlated residuals between GAD symptoms and perceived attachment relationship quality with fathers. These correlations were stronger for boys (ß’s ranged from −0.12 to −0.20) than for girls (ß’s ranged from −0.05 to −0.17).χSB²  = 21.64, p < 0.05. The models with different paths from GAD symptoms to perceived attachment relationship quality, ΔχSB²  = 0.74, p = 0.39, and with different paths from perceived attachment relationship quality to GAD symptoms, ΔχSB²  = −0.11, p = 1.00, did not provide a better fit. Model fit for the final model for fathers was: (34) = 212.96, p = 0.66; CFI = 0.94; RMSEA = 0.09.Using multigroup analyses with the two age cohorts as the two groups, we tested whether the within-wave correlated residuals and cross-lagged paths between GAD symptoms and perceived attachment relationship quality could be fixed across groups. For perceived attachment relationship quality with fathers, the chi-square difference tests showed that the model with different paths for the two age cohorts provided a better fit to the data than the model with equality constraints across groups for within-wave correlated residuals, ΔχSB²  = 0.45, p = 0.80, and for paths from GAD symptoms to perceived attachment relationship quality, ΔχSB²  = 2.80, p = 0.09. Model fit for the final model for mothers was: χSB² (41) = 217.02, p = 0.35; CFI = 0.94; RMSEA = 0.08.For adolescents’ perceived attachment relationship quality with mothers, the model with different paths for the groups did not provide a better fit for the data than the model with equality constraints across groups for within-wave correlated residuals, ΔThus, the only difference between the early and middle adolescents cohort was in the within-wave correlated residuals between GAD symptoms and perceived attachment relationship quality with fathers. These correlations were stronger for the cohort of middle adolescents (ß’s ranged from −0.10 to −0.19) than for the cohort of early adolescents (ß’s ranged from −0.07 to −0.17). Figures The main aims of this study were to investigate the bidirectional relation between perceived attachment relationship quality with both fathers and mothers and GAD symptoms over time, and to examine whether these associations differed for boys and girls, and early and middle adolescents. The results showed a bidirectional relation between GAD symptoms and perceived quality of attachment relationship with fathers, whereas a unidirectional model was found for mothers. Only adolescents’ GAD symptoms longitudinally predicted perceived mother-adolescent attachment relationship quality. Gender and age moderated the within-wave correlated residuals between GAD symptoms and attachment relationship quality with fathers, but not with mothers.The most consistent evidence was found for child effects: Adolescents who reported higher levels of GAD symptoms perceived lower quality of attachment relationship with both fathers and mothers one or two years later. This finding can be interpreted in two ways. A possibility is that parents of adolescents with self-reported GAD symptoms change their behaviors toward their adolescent children. It is assumed that emotional problems of adolescents could lead to negative responses of their parents (Bögels and Brechman-Toussaint In addition to the child effects, parent effects were found for the attachment relationship with fathers, but not for the attachment relationship with mothers. Adolescents who perceived lower quality of attachment relationship with fathers reported higher levels of GAD symptoms one or two years later. This finding supports attachment theory that proposes that parents foster healthy development among adolescents Bowlby . In addiAlthough we found a difference between GAD and attachment relationship quality with fathers and mothers, it was not the difference that was expected. We hypothesized that the relation between GAD symptoms and perceived attachment relationship quality would be found for mothers but not for fathers Liu , yet finBesides the longitudinal relation between GAD symptoms and perceived parent-adolescent attachment relationship quality, there were also within-wave correlated residuals between those constructs. In accordance with the findings of Hale et al.  and VianWe investigated whether the relation between perceived attachment relationship quality with fathers and mothers and GAD symptoms differed for boys and girls. We expected that this relation would be stronger for girls than boys, because girls have a stronger relational orientation Rudolph  and a geBesides adolescent gender differences, we also examined the moderating role of age. No differences were found between the cohort of early adolescents and the cohort of middle adolescents for the cross-lagged paths. Age only moderated the within-wave correlated residuals between adolescents’ GAD symptoms and perceived father-adolescent attachment relationship quality. Contrary to what was expected, we found that the correlations for middle to late adolescents were slightly stronger than for early to middle adolescents. This finding can possibly be explained by the fact that GAD symptoms in adolescence increase (Hale et al. The current study has several important strengths. Firstly, we extended current knowledge of the relationship between adolescents’ GAD symptoms and perceived attachment relationship quality by using a longitudinal design. This allowed us to disentangle the direction of paths between GAD symptoms and perceived parent-adolescent attachment relationship quality. Previous studies investigating this association were cross-sectional. Many studies still rely on a parent effect model, but our study confirms that it is important to take account of child effects. Furthermore, the relation between GAD symptoms and perceived attachment relationship quality was examined separately for fathers and mothers. This is an important strength as fathers are generally neglected in research on adolescent anxiety (Bögels and Phares Several limitations should also be mentioned. First, the present study only included adolescents from the general population. Because of this, our study is not comparable with studies that gathered data from adolescents diagnosed with GAD. However, research on this population can give insight into issues relevant to the clinical setting. Moreover, since the sample consisted predominantly of Dutch adolescents, the results of the present study cannot be generalized to other samples of adolescents. Furthermore, although the longitudinal design of our study gives insight in the direction of effects, no causal conclusions can be drawn. Another caveat of the current study is the use of adolescents’ self-reports to assess the quality of the parent-adolescent attachment relationship. It is not certain that parents’ actual behavior exactly corresponds to the adolescent’s perception of parental behaviors. Additionally, parents’ and adolescents’ perceptions of the quality of their attachment relationship often diverge (Renk et al. In sum, the results of the present study indicate that adolescents’ GAD symptoms and perceived parent-adolescent attachment relationship quality are longitudinally associated. The direction of effects depends on parents’ gender. GAD symptoms consistently affected later perceived attachment relationship quality with both parents. Only for fathers, the perceived quality of attachment relationship predicted later GAD symptoms. However, child effects were stronger. Furthermore, our results revealed that the within-wave correlated residuals between GAD symptoms and perceived father-adolescent attachment relationship were stronger for boys than for girls, and stronger for middle adolescents than for early adolescents. So, there is a longitudinal, negative relation between GAD symptoms and perceived parent-adolescent attachment relationship that depends on the interplay between parent and adolescent gender an adolescent age. Prevention and treatment programs for GAD symptoms should focus on how these symptoms affect adolescents’ perception of the attachment relationship quality with parents. Moreover, our results show the importance of encouraging paternal involvement in adolescents, as well as the importance of involving fathers, in addition to mothers, in research and treatment of GAD symptoms in adolescence.


The Tasmanian Devil Facial Tumour Disease (DFTD) provides a unique opportunity to elucidate the long-term effects of natural and anthropogenic selection on cancer evolution. Since first observed in 1996, this transmissible cancer has caused local population declines by >90%. So far, four chromosomal DFTD variants (strains) have been described and karyotypic analyses of 253 tumours showed higher levels of tetraploidy in the oldest strain. We propose that increased ploidy in the oldest strain may have evolved in response to effects of genomic decay observed in asexually reproducing organisms. In this study, we focus on the evolutionary response of DFTD to a disease suppression trial. Tumours collected from devils subjected to the removal programme showed accelerated temporal evolution of tetraploidy compared with tumours from other populations where no increase in tetraploid tumours were observed. As ploidy significantly reduces tumour growth rate, we suggest that the disease suppression trial resulted in selection favouring slower growing tumours mediated by an increased level of tetraploidy. Our study reveals that DFTD has the capacity to rapidly respond to novel selective regimes and that disease eradication may result in novel tumour adaptations, which may further imperil the long-term survival of the world's largest carnivorous marsupial. More than 35 years ago, Nowell  suggesteThe presence of aberrant karyotypes in malignant cells was first observed over a century ago (von Hansemann Sarcophilus harrisii) by biting during social interactions  was added, and the tubes were centrifuged for 10 min at 100 g. The pellet was gently resuspended in fixative and stored at −2°C overnight. The following day, the cells were washed 4× in fresh fixative and resuspended. Chromosome spreads were achieved by adding a droplet of the suspension onto a frozen microscope slide. Slides were subsequently air-dried and incubated at 57°C for 3 days. G-banding was conducted by treating slides with a 0.15% solution of trypsin (Sigma-Aldrich) for 30 s, then staining with Leishman's stain for 2.5 min followed by mounting with Leica mounting medium  for analysis. G-banding analysis was performed using a Leica DM 2000 microscope (Leica Microsystems) and photographed with a Leica DFC 420 C camera (Leica Microsystems). Karyotypes were made, originally by hand and later (from 2008) using Video testeKaryo 3.1 software . At least 20 metaphases were analysed for each individual, and approximately, 200 cells were examined.Three hours prior to harvesting 0.1 mL of demecolcine at 10 mg/mL (Sigma-Aldrich) was added to each culture. The cells were centrifuged for 10 min at 100 Data were examined for normality before analysis, and when normality could not be achieved, nonparametric statistics were employed. Logistic regression was used when investigating temporal variation in tetraploidy among the 253 devil tumours where presence, that is, when all of 200 cells in the metaphases were recorded as being tetraploid, was entered as ‘1’ and total absence of tetraploids was entered as ‘0’. JMP, version 5.1 , were used in all statistical analyses.2 = 11.7, P = 0.008, df = 3 and χ2 = 9.6, P = 0.02, df = 3, respectively, The large number of karyotypes used in this study made it possible to conduct robust tests to elucidate a possible variation in tetraploid tumours among the four strains. Our results revealed a significant variation of tetraploid tumours when employing the total number of samples collected at the 11 sites as well as when omitting the samples collected at the Forestier peninsula (χ2 = 16.4, P < 0.0001, df = 1; 2 = 0.15, P = 0.70, df = 1; 2 = 35.0, P < 0.0001, df = 1; We also observed a significant temporal increase in tetraploid tumours collected at the 11 sites from 2006 to 2011 (logistic regression with tetraploidy as dependent and year as independent variable: χin vivo.Devil Facial Tumour Disease is a horizontally transferred asexually reproducing clonal cell line, which during the last 16 years have been exposed to the negative effects associated with Muller's ratchet, resulting in mutational meltdown and ultimately extinction. However, this obligate parasite has been able to survive and counteract the effect of deleterious mutations, genomic instability as well as being able to infect >100 000 devils McCallum . DFTD hePolyploidization is more common in asexual compared to sexual organisms (Otto and Whitton In a recent study, Murchison et al.  suggesteTransition from whole-genome duplication via aneuploidy to malignancy is a common feature of several human cancers (for review see Davoli and de Lange


Background: Cardiac output (CO) measurement in low (LBW) and very low (VLBW) birth weight infants is difficult. Hitherto, sporadical transthoracic echocardiography (TTE) is the only non-invasive measurement method. Electrical velocimetry (EV) has been evaluated as an alternative in normal weight newborns.Objectives: The study was designed to evaluate if EV could be interchangeable with TTE even in LBW and VLBW infants.Methods: In 28  pre-mature newborns, n = 228 simultaneous TTE (trans-aortic Doppler), and EV measurements  of stroke volume (SV) and heart rate (HR) were performed, thereof calculating body weight indexed SV (=SV*) and CO (=CO*) for all patients and the subgroups. Method comparison was performed by Bland–Altman plot, method precision expressed by calculation of the coefficient of variation (CV).Results: Mean CO* in all patients was 256.4 ± 44.8 (TTE) and 265.3 ± 48.8 (EV) ml/kg/min. Bias and precision were clinically acceptable, limits of agreement within the 30% criterion for method interchangeability (eability . AccordiConclusion: Extending recent publications on EV/TTE comparison in newborns, this study suggests that EV is also applicable in LWB/VLBW infants as a safe and easy to handle method for continuous CO monitoring in the NICU and PCICU. Information about cardiac output (CO) in newborn and especially in pre-term, low birth weight (LBW), and very low birth weight (VLBW) infants is difficult to obtain. However, this information is particularly important in such patients with their numerous circulatory features  and EV TTE, SVEV, Descriptive statistics for SVTTE and SVEV were significantly lower in VLBW patients compared to those in the LBW group due to their smaller anatomic dimensions (p < 0.001) whereas there was no statistically significant difference between VLBW and LBW infants for TTE and body weight was r = 0.77 and between SVEV and body weight r = 0.81.As could be expected, SVp = 0.009 for p < 0.001).EV 2.88 ± 0.83 ml, EV 2.51 ± 0.41 ml, p = 0.002, p = 0.031). HR was significantly in patients without (163.5 ± 9.1 beats/min) than with inotropic treatment . Consequently, CO* was significantly higher in these patients, too (p = 0.009).SV and SV* were higher in patients with inotropic treatment (SVp = 0.024) based on a higher SVEV .For all patients, This study was a pure observational method comparison of EV and TTE in LBW and VLBW infants with supplementary clinical observations indicating a possible use of EV in daily neonatological and cardiological practice. It was not designed to evaluate a “true” CO comparing EV to a convened gold standard like Fick’s method or thermodilution, which is not possible in very small infants, but to explore the utility of EV as an easily feasible alternative to the technically demanding trans-aortic Doppler for the measurement of CO in these patients.Method comparison of the two non-invasive CO measurement methods, TTE and EV, shows that they are interchangeable, fulfilling the criteria of Critchley and Critchley , with naMethod interchangeability was not altered by the respiratory mode (ventilation or spontaneous respiration) whereas Stroke volume correlated well with the patients’ body weight and was therefore higher in LBW than in VLBW patients, thus reflecting correctly the patients’ differences of weight and age. As the body weight related SV* was not different in the two groups, the paradoxically higher CO* in the VLBW group was obviously due to a higher HR in these patients.The higher SV and CO in ventilated patients may be due to positive effects of positive intrathoracic pressure on left ventricular (LV) afterload reduction as has been previously reported , 2.Extending the previous studies in normal weight newborns , 16, theAs a conclusion, EV may represent a valuable tool for CO monitoring in LBW and VLBW infants, particularly helpful in detecting imminent risks of low CO  associatEV, Compared to TTE, the advantage of EV may be a continuous and relatively easy manageable CO monitoring completing the “traditional” circulatory monitoring in neonatal, pediatric, and pediatric cardiological intensive care units. Its clinical utility still needs to be confirmed, but evidence is growing , 16. In The Review Editor Yves Durandy declares that, despite being affiliated to the same institution as author Oswin Grollmuss, the review process was handled objectively and no conflict of interest exists. The other co-author reports no conflicts of interest.