Electroencephalography studies in schizophrenia reported impairments in circadian rhythm and oscillatory activity, which may reflect the deficits in cognitive and sensory processing. The current study evaluated the circadian rhythm and the state-dependent oscillatory pattern in control Wistar and a multiple hit schizophrenia rat model (Wisket) using custom-made software for identification of the artifacts and the classification of sleep-wake stages and the active and quiet awake substages. The Wisket animals have a clear light-dark cycle similar to controls, and their sleep-wake rhythm showed only a tendency to spend more time in non-rapid eye movement (NREM) and less in rapid eye movement (REM) stages. In spite of the weak diurnal variation in oscillation in both groups, the Wisket rats had higher power in the low-frequency delta, alpha, and beta bands and lower power in the high-frequency theta and gamma bands in most stages. Furthermore, the significant differences between the two groups were pronounced in the active waking substage. These data suggest that the special changes in the oscillatory pattern of this schizophrenia rat model may have a significant role in the impaired cognitive functions observed in previous studies. Schizophrenia is a chronic and multidimensional neuropsychiatric disorder with devastating consequences for patient outcomes. Although not included in its diagnostic criteria, sleep disturbances are consistently reported in patients, including disrupted circadian rhythms of activity, sleep-wake, and oscillatory patterns obtained by electroencephalography (EEG) . All theRodent models are essential for understanding brain function in health and disease. Several animal models of schizophrenia have abnormalities in their circadian rhythm and oscillatory pattern, but most of these results obtained in mutant mice with modification of only one gene transcription, e.g., disrupted-in-schizophrenia 1 (DISC1) gene, metabotropic glutamate 5 receptors (mGLUR5), voltage gated L-type calcium channels, serine racemase enzyme (responsible for N-methyl-D-aspartate (NMDA) receptor function), GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-izoxazolepropionic acid (AMPA) receptors, or stable tubule only polypeptide (STOP) . Only fe2R and D1R) signaling and/or expression , pharmacological , and genetic  manipulations, as described previously , 2021a. pression . Furtherpression , 2016. Tvia visual inspection of the EEG features, which is a laborious task prone to interindividual variability and it also requires considerable time and specialized knowledge about sleep in rodents. Considerable progress in computational technologies has made possible an improvement in automated sleep scoring algorithms  and Wisket rats (4–6 months old) were involved in the study. All experiments were carried out with the approval of the Hungarian Ethical Committee for Animal Research (registration number: XIV/1248/2018) and in accordance with the guidelines set by the Government of Hungary and EU Directive 2010/63EU for animal experiments. The animals were kept with a 12 h light/dark cycle under conditions of controlled temperature (23 ± 1°C) and humidity (55 ± 10%).The whole setup of the surgery was similar as described previously . Brieflyad libitum. The room was sound attenuated. During this period, the rats were connected to the recording tether and habituated to the experimental conditions. After the recovery period, EEG was recorded for 23 h . However, due to technical reasons , 21 h-long EEG recordings were analyzed, including the total length of the light phase (12 h), and 9 h of the dark phase.After the surgery, the rats were housed individually in custom-made clear plexiglass cages (25 cm × 28 cm × 50 cm), to avoid the removal of the implanted devices by their cage mate. The rats were in visual, auditory, and olfactory contact with other rats to minimalize the effect of isolation rearing . They wen = 8/group) were recorded simultaneously in each recording session. Furthermore, to determine whether the changes obtained between the two groups in the first series can be reproduced independently of the environmental circumstances, the second series of experiments was performed with a different group of animals, where the animals were unmatched .Two series of experiments were performed. In the first (matched) series, four rats  and filtered (0.5−48 Hz), and were digitized by an inbuilt analog-to-digital converter at a sampling rate of 128 Hz. The resolution was 12 bits. The motor activity of the rats was detected by a 3 − axis accelerometer  within the unit, as described earlier , 2004. FThe EEG recordings contained artifacts, caused by short contact losses of the electrodes due to animal movements. During these periods, the EEG signal clipped and these sections’ spectra would mislead the spectrum-analysis approach presented in the article. To correct these sections, the EEG signals were preprocessed. First, the clipping signal parts were detected, and then they were analyzed and substituted. The replacement signal had the same length as the faulty section and was generated as a well-formed (colored) white Gaussian noise, which noise’s spectrum was formed to match the spectrum of the EEG signal surrounding the faulty section. Thus, the replacement does not affect the aggregated spectrum-based features extracted at the later stages of the analysis. The preprocessing method corrected 2% of the EEG dataset.The next task was to classify the EEG signal into three wake-sleep stages: awake, non-rapid eye movement (NREM), and rapid eye movement (REM) stages. To achieve this automated classification, a machine-learning model was trained to predict the class label for 8 s-long EEG signal segments. To train such a model, we had access to a dataset containing 36 manually classified recorded days and approximately 10, 000 hand-labeled segments for each day. Our approach was to extract handcrafted features from the segments and use only these to predict a label. The generated features are the following: mean amplitude and SD values for the different frequency bands, the SD of the motion data, and a Petrosian Fractal Dimension value  of the EAfter the categorization of the states of vigilance, power spectra were computed by Fast Fourier Transform (FFT) for each stage and substages under a condition of 0.125°Hz resolution with a Hanning window. Total absolute power was calculated as the sum of squares of all frequency values contained in a given band. Relative band powers were expressed as power ratios of each frequency band to the total (z-score) in each 8 s bin. The resulting power spectra were divided and analyzed in different frequency ranges: delta (0.5−4 Hz), theta (4−8 Hz), alpha (8 − 12 Hz), beta (12 − 30 Hz), and low gamma (30–48 Hz) bands.The parameters were quantified and analyzed as means of values per hour for the 21 h (daily) and separately for the light and dark phases . The mean duration per hour and the number and duration of episodes in different stages were determined. Regarding the detailed analysis of the oscillatory pattern, our preliminary data analysis revealed a separate or even opposite tendency in relative powers in narrow frequency ranges. Therefore, based on earlier results, we performed the statistical analysis for all of the data within a frequency band  to reveal fine-grained alterations between the two groups .post-hoc test was used for the evaluation of the effects of various parameters. Statistical analysis was performed with STATISTICA 13.5.0.17 . Differences were considered significant for p < 0.05.Data are expressed as means ± SEM. The parameters were compared with repeated and/or factorial ANOVA. When the global test was significant, the Fisher least significant difference (LSD) F = 282.62; p < 0.001; F = 29.82; p < 0.001], thus, both groups of animals spent less time in quiet awake than in active awake substage  = 268.94; p < 0.001) and phase and stage interaction , phase , and substage and phase interaction , thus both groups of animals spent a long time in both types of awake substages during the dark than in the light phase  during the whole investigated period showed significant effects of the stage  and phase , thus, both groups of animals had fewer number in bouts of all stages during the dark phase, with a moderate tendency of decreased numbers in Wisket animals  = 24.75; p < 0.001], phase , and the stage and phase interaction , thus, both groups of animals had shorter REM sleep episodes compared to awake and NREM stages, and longer awake bouts during the dark phase .The oscillatory pattern of the EEG was analyzed next. Since no diurnal changes were obtained in the oscillations, power spectra were analyzed for the whole session independently of stages, and then for the three sleep-wake stages  and the active and quiet awake substages, separately. To demonstrate the fine-grained distribution of the relative power, the ANOVA analysis was performed for all the data obtained within the different frequency bands .Regarding the analysis of total absolute power, only a tendency of enhanced power was obtained in the Wisket animals by stages, substages, and the light-dark phases .F = 157.82; p < 0.001] and group and frequency interaction . The post hoc analyses revealed that the Wisket animals had significantly higher total relative power compared to control ones in the interval of 1.38 − 1.75 Hz (Regarding the entire relative power irrespective of the stages in the delta band (0.5 − 4 Hz), factorial ANOVA showed significant effects of frequency , stage , group and frequency interaction , group and stage interaction , frequency and stage interaction , and group, frequency, and stage interaction . The post hoc analysis disclosed that the delta power was the lowest during the REM stage in both groups, with significantly lower in its middle range (1.88 − 2.5 Hz) in the model rats compared to control animals. Furthermore, the pattern of the delta spectrum differed between the two groups in the NREM stage, i.e., the Wisket animals had higher power at the lower frequency range (1.25 − 2 Hz), while they had significantly lower power at the higher frequency range (3 − 4 Hz) compared to control rats  = 16.75; p < 0.001], frequency , substage , and frequency and substage interaction . Thus, the Wisket animals had a trend for higher delta power in both awake substages  = 10.58; p < 0.001] and frequency . The post hoc analysis showed that the Wisket animals had significantly lower power compared to control ones at the higher frequency theta band , factorial ANOVA showed significant effects of group , frequency , stage , group and frequency interaction , group and stage interaction , frequency and stage interaction  and group, frequency, and stage interaction . Thus, the pattern of the theta spectrum differed between the stages with the lowest rise of the curve during NREM  = 123.00; p < 0.001], frequency , substage , group and frequency interaction , group and substage interaction  and frequency and substage interaction . Thus, the Wisket animals in both active and quiet substages had significantly lower total power  = 52.00; p < 0.001] and frequency . The Wisket animals had significantly higher relative power compared to control ones at the lower range of alpha band , factorial ANOVA showed significant effects of group , frequency , stage , group and frequency interaction , group and stage interaction , frequency and stage interaction  and group, frequency, and stage interaction . The pattern of the alpha spectrum differed between the stages with the lowest rise of the curve during NREM, and the Wisket animals had higher relative alpha power in awake and REM stages in the lower frequency bands  compared to control rats  = 126.58; p < 0.001], frequency , group and frequency interaction , group and substage interaction , and frequency and substage interaction . The post hoc comparison revealed that the Wisket animals had significantly higher relative power at the lower frequency alpha band (8.38 − 9.75 Hz) compared to controls during the active awake substage  = 344.18; p < 0.001], frequency , and group and frequency interaction . Thus, the Wisket animals had significantly higher total relative power compared to control ones at the lower frequency beta band  = 625.89; p < 0.001], frequency , stage , group and stage interaction , and frequency and stage interaction . The pattern of the beta spectrum differed between the stages with the highest steepness of the curve during NREM, and the Wisket animals had higher relative power in all stages at the lower beta frequency band  = 541.50; p < 0.001], frequency , substage , group and frequency interaction , and frequency and substage interaction . Thus, the Wisket animals had significantly higher relative power at the lower frequency beta band compared to controls primarily during the active awake substage  = 588.00; p < 0.001) and frequency , frequency , stage , and frequency and stage interaction . The post hoc analysis showed that the pattern of the gamma spectrum differed between the stages with the lowest steepness of curve values during NREM, and the Wisket animals had lower relative gamma power, primarily during the awake stage at the higher frequency range  = 478.00; p < 0.001], frequency , substage , group and substage interaction , and frequency and substage interaction . Thus, the Wisket animals had significantly lower relative power at the higher frequency gamma band compared to controls, primarily during the active awake substage  compared to the controls. However, the Wisket animals showed significantly enhanced delta activity in its lower frequency range during the NREM phase, while it decreased in the higher frequency range of the delta band during the NREM and REM stages. The higher power in the lower frequency delta band might suggest enhanced requirements of sleep . The redThe cortico-hippocampal circuits are key generators of theta rhythm, which has an important role in cognition and memory formation . Theta oInconsistent changes in theta band power were observed in patients with schizophrenia  dependinIn Wisket rats, the significantly lower relative power of the higher frequency band was observed during the awake (both the active and quiet substages) and REM stages. In contrast, they had significantly lower power at the lower frequency range of theta band during NREM. Optogenetic inhibition of REM theta power impairs sleep-dependent memory consolidation in mice . TherefoThe oscillation in the alpha band is related primarily to the thalamus, thus, the alterations in this frequency band may suggest dysfunction of the inhibitory input of thalamic neurons . There aWhile Wisket animals showed significantly enhanced relative power at the lower frequency alpha band during awake (primarily in active substage) and REM stages, it might be related to the negative symptoms. In agreement with our results, hippocampal lesion increased the alpha power during the awake stage , but AMPBeta oscillations are believed to be generated broadly across multiple neocortical structures and are involved in adaptation to repetitive sensory stimuli, attention, affective processing, alertness, and synchronization of large ensembles of neurons . Both deOur data clearly showed that Wisket animals had significantly enhanced beta power at the lower frequency range in all stages compared to control animals, but this difference was most prominent during active awake substage. These alterations might play role in the impaired cognitive behavior of Wisket model rats observed earlier . PreclinGamma oscillations have received great interest because of their role in sensory and cognitive functions such as selective attention, short- and long-term memory, and multisensory integration . The priOur results showed that Wisket animals had a significant decrease in the relative power of the higher band of gamma wave (between 39 and 48 Hz) compared to the controls, which was dominant during the awake stage (primarily in the active substage). It is well-known that gamma band oscillations are associated with multiple cognitive functions, and gamma activity increases during alert or attentive wakefulness, especially above 40 Hz . TherefoThere are several limitations in our recording system: the maximum recording duration and the limited number of channels, and a lack of deep hippocampal electrodes. Furthermore, as we used a tethered option rather than wireless recording, the rats were singly housed in cages, to provide appropriate circumstances for fully recovering after the surgery and prevent the destruction of the EEG devices and cables. It is well known that long-term single housing (more than 1 week), especially in young animals, affects several biological parameters . HoweverOur data first characterized the sleep-wake rhythm and oscillatory pattern of a multiple hit rat model of schizophrenia. The results indicate that the Wisket rats have only a tendency to alterations in their sleep-wake pattern with normal circadian rhythm. On the other hand, the investigation of the oscillatory patterns in different frequency bands revealed complex alterations in all stages and substages of the sleep-wake rhythm.Wakefulness and sleep are promoted by the ascending arousal and descending inhibitory pathways, respectively, operating with several neurotransmitters, including dopamine, glutamate, and GABA systems . The WisA better understanding of the characteristics of sleep disturbance and oscillatory activity in this schizophrenia model may help to address fundamental relationships among the behavioral, neurochemical, and electrophysiological parameters, as well as to develop novel drug targets and therapies to treat this disorder.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Hungarian Ethical Committee for Animal Research.LA contributed to data collection, interpretation of the results, and manual classification. MC-N developed data classification software and tested by comparison with manual classification. BB did animal surgery, conducted the experiments, and developed the data recording system. GyK developed data classification software and contributed to statistical analysis. LN supervised software development and performed proofreading. AN supervised the experiments and manual classification of data and performed proofreading. GaK contributed to the interpretation of the results and performed proofreading. AB contributed to statistical analysis. GH conceived of the presented idea, planned the experiments, performed statistical analysis, and wrote the first draft of the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pygoscelis papua), chinstrap (P. antarcticus), and Adélie penguins (P. adeliae) (n = 108) were collected by passive sampling in seven different locations throughout the Antarctic Peninsula area and analyzed by ICP-MS after microwave-digestion. More than 93% of the samples showed detectable Hg levels , and the highest ones were found in the feathers of chinstrap penguins from King George Island. Hg bioconcentration and biomagnification seem to be occurring in the Antarctic food web, giving rise to high but non-toxic Hg levels in penguins, similar to those previously found in Arctic seabirds.Polar regions, symbols of wilderness, have been identified as potential sinks of mercury coming from natural and anthropogenic sources at lower latitudes. Changes in ice coverage currently occurring in some areas such as the Antarctic Peninsula could enhance these phenomena and their impacts on local biota. As long-lived species at the top of food chains, seabirds are particularly sensitive to this highly toxic metal with the capacity to be biomagnified. Specifically, their feathers can be useful for Hg monitoring since they mainly accumulate its most toxic and persistent form, methyl-Hg. To that end, feathers of gentoo ( It has been reported that each change in the food chain accounts for an increase of an order of magnitude in methylmercury concentrations [Mercury levels naturally present in the environment have increased during the last decades as a consequence of direct emissions from human activities  and the mobilization of previously deposited mercury ,2. This trations . As a cotrations . MoreoveAmbient monitoring allows us to know the levels of environmental chemicals present in different environmental compartments , but only the analysis of these chemicals in wildlife can offer a real and precise picture of the amount of chemicals that is really absorbed by them, which therefore exerts adverse effects on health. Biological monitoring, i.e., the analysis of chemicals or metabolites in biological samples, is therefore a useful tool to investigate the body burden and potential health effects of mercury in wildlife.The toxic effects of this pollutant on wildlife have been studied in different ecosystems, which are varied and complex and include physiological, neurological, behavioural, and reproductive harm causing chromosomal alteration, endocrine disruption, or effects on the immune system, among other impacts ,8,9.In this context, and although Antarctica can be considered one of the few environments still unspoiled worldwide, specific incidents and the increasing tourism, together with global pollution, are increasing contamination and adding pressure to the equilibrium and conservation of this unique ecosystem ,11. TherIn general, blood is considered the best matrix for biological monitoring of the exposure to the majority of chemicals . HoweverPygoscelis papua), chinstrap (P. antarcticus), and Adélie (P. adeliae), from seven different locations in a NE–SW gradient from high to low human activity throughout the Antarctic Peninsula area. Potential higher levels of mercury in the northern locations, which have the higher human impact [This study investigated the exposure to mercury in three species of penguins, gentoo  were captured during moulting using a long-handled net, sampled, and immediately released. The sample size for each location and species is shown in During the 2005/2006 and 2006/2007 austral summer seasons, feather samples  were collected from the flanks of adult penguins in seven different locations throughout the Antarctic Peninsula area and individually stored in polyethylene bags. Penguins (3 (65%), H2O2 (30%), and H2O in the proportion of 5:2:3. Hg concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS Agilent 7500 ce). The system included a CETAC ASX-510 autosampler, a Peltier-cooled Scott-type nebulizer chamber, a MicroMist concentric nebulizer, nickel cones, a 27.12 MHz radio frequency generator, a 1600 W Fassel-type quartz torch, an argon mass flow control in plasma, an auxiliary line, an adjustment line and carrier gas, a hyperbolic quadrupole mass filter (3 MHz and 2–260 amu), a simultaneous digital/analog detector with 9 orders of magnitude of linear dynamic range, and a collision/reaction cell.Samples were analysed using the analytical method described by Jerez et al.  with min3 (10%) for 24 h, including a last rinsing step with double-distilled and deionized water, final ultrasonic cleaning, and subsequent oven-drying (70 °C). All reagents used were Suprapur (Merck), and the water was double-distilled and deionized . Quality control of the procedure (reproducibility and reliability of the results) was carried out by introducing the samples in duplicate at random, with blanks at the beginning of each series of analyses and at every 5 samples. The calibration standards were analyzed initially and periodically. Certified reference materials were used , which were stabilized with 20 μL of HNO3. The concentrations of the standards were 1, 5, 10, 25, 50, 100, and 200 ng/L. In cases where the upper limit of the calibration line was insufficient to detect levels higher than this in the test samples, standards of a higher concentration or dilution of the sample in question were prepared. The correlation index (R2) of the calibration lines was equal to or greater than 0.999.In order to avoid contamination, all the materials used were carefully washed in HNOThe detection limit was calculated by using the formula DL = 3 sB/a . According to Smith et al. , values t-tests. All statistical analyses were two-tailed with a significance level of 0.05. Inter-specific differences were tested in those locations where two or three species shared the same area, whereas geographical differences were tested for each species separately.Statistical analyses were conducted using Statistica package version 7. Variables were normally distributed (Kolmogorov–Smirnov test > 0.20). Inter-specific and geographical differences were tested by using one-way ANOVAs (with Bonferroni post-hoc tests) and Student′s P. papua), chinstrap (P. antarcticus), and Adélie penguins (P. adeliae) and the study of geographical differences are shown in P. papua and P. adeliae and in the 82% of the samples of P. antarcticus. As mentioned before, mercury present in feathers was mainly in the form of methyl-Hg (percentages higher than 80% in aquatic bird feathers) [Concentrations of Hg in the feathers of gentoo (eathers) , so the −1. The greater proximity of King George Island to South America, and therefore to anthropogenic sources of mercury, as well as a higher human activity intensity in this location could explain this result; although, 70% of samples in this location showed mercury levels below the detection limit (LD). On the contrary, the results for Adélie penguins seem to support our prediction as the highest levels were found in King George Island; although, differences with the other sampling sites were only marginally significant  [Hg concentrations in penguin feathers in this study were four to six orders of magnitude higher than those found in inorganic samples such as snow from different Antarctic areas . This id et al. . Besides−1 d.w.; ,32). Thew.; [−1) .−1 d.w. [−1 d.w.), although large inter-individual differences were observed, which could perhaps be influenced by the size or age of the individuals [−1 d.w. [Comparing our results with those described in the feathers of other seabirds from different regions, we found that some chinstrap penguins from King George Island showed high Hg levels, similar to those found in Arctic seabirds . Theividuals . The oth[−1 d.w. ,36,37). [−1 d.w. .High Hg levels found in penguin feathers confirm the idea that, during spring and summer, the coastal Antarctic ecosystems can be affected by similar or higher Hg deposition rates than many other regions in the world. Although this phenomenon has been more widely studied in the Arctic, some studies have also confirmed the existence of Hg depletion events in Antarctica ,39 as wehttp://www.mercuryconvention.org/ (accessed on 17 July 2021)). However, despite the current efforts to reduce the use of mercury and protect the environment as well as human populations, the re-emission and the long-range atmospheric transport of this element make it that mercury levels in the environment will not decrease on a short time scale. According to Bargagli [Today these processes achieve special relevance in Antarctica because the use of Hg, among other pollutants, is increasing in the Southern Hemisphere and Asia, contrary to what is happening in North America or Europe. Mercury in the environment is a global problem that requires global solutions such as the Minamata Convention , they can also be related to anthropogenic pollution in the region. The latter would explain the high Hg levels detected in the feathers of chinstrap and Adélie penguins collected in King George Island, one of the Antarctic locations with greater human activity and the nearest to South America. This fact could be especially relevant in top predators such as penguins, since the results support the existence of mercury bioconcentration and biomagnification in the Antarctic trophic chain.Although the levels detected in this study are lower than those considered toxic for seabirds, they are similar or even higher than those found in seabirds from other regions of the world, supporting the hypothesis of mercury depletion, bioaccumulation, and biomagnification in Polar Regions.Since climate change and rising temperatures can exacerbate the situation by increasing mercury atmospheric concentrations and methylation, this could increase the risk for wildlife in this unique and mercury-sensitive environment. Thus, the control of these pollutant levels present in the Antarctic fauna, and especially in top predators, is crucial for monitoring potential adverse effects.Penguin feathers are a biological matrix that is particularly useful for monitoring Hg levels and investigating how diet and other factors are related to the levels found; they also support a better understanding of spatial and temporal trends.


Viola L. In most sections of Northern Hemisphere violets, cleistogamy is seasonal, and CH and CL flowers develop sequentially in the season. Non-seasonal cleistogamy (simultaneous) is a rare phenomenon in rostrate violets. In the current study, we focused on modification of the CH/CL mating system in V. caspia by environmental conditions, resulting in a gradual switch from temporal cleistogamy, occurring in nature, to simultaneous cleistogamy under greenhouse conditions. V. reichenbachiana with seasonal cleistogamy was the control for V. caspia with the labile seasonal/simultaneous cleistogamy system. In simultaneous cleistogamy, the CH and CL flowers, fruits and seeds developed on an individual plant at the same time on the same branch. The typical difference between CH and CL flowers’ pistils is a straight style ending with a head-like stigma in CH and a curved style in CL adapted to self-pollination. This trait persists in the fruit and seed stages, allowing for easy recognition of fruit of CL and CH flowers in simultaneous cleistogamy. Floral meristems of CH flowers of V. reichenbachiana developed on the rhizome at the end of the growing season under short-day conditions and remained dormant until the following season. The CL floral meristems formed under long-day conditions on elongating lateral branches in the upper leaf axils. The daily temperature influenced the variable CH/CL ratio of V. caspia in nature and greenhouse conditions. Regulation of the CL/CH flower ratio by modifying environmental factors is important for basic research on genetic/epigenetic regulation of cleistogamy and for practical use to produce genetically stable lines of economically important species via CL seeds.The special mixed reproductive system, i.e., the ability of an individual plant to develop both open, chasmogamous (CH) flowers adapted to cross-pollination and closed, cleistogamous (CL) flowers with obligate self-pollinating, is a common phenomenon in  Viola species . Induction of CL flower development is important in crops  and medicinal and ornamental plants to produce genetically uniform selected lines through self-pollination. The physiology, genetic/epigenetic regulation and molecular mechanisms of cleistogamy still need extensive investigation, although several papers have been published over the last decades [The interest in cleistogamy research is mostly focused on the morphology of CH and CL flowers, factors inducing CL flowering and the genetic and molecular basis of the development of both flower types. Different heterochronic processes are involved in the formation of small, unopened CL flowers in ve to CH ,13,14. T decades ,21,22,23Viola, the largest genus in the family Violaceae, comprising ca. 600 species distributed mainly in the Northern Hemisphere [Viola sections are characterized by seasonal cleistogamy in which CH and CL flowers do not develop simultaneously on an individual plant, but temporal production of CH and CL flowers is strongly influenced by environmental factors such as light quantity, canopy cover, photoperiod, temperature, soil pH and moisture [Cleistogamy is relatively common in misphere ,25, whicmisphere ,26,27,28moisture . V. reichenbachiana Jordan ex Bor. and V. caspia (Rupr.) Freyn, are caulescent perennials belonging to the palaeotetraploid sect. Viola subsect. Rostratae (Kupffer) W. Becker [Viola reichenbachiana is distributed in western Eurasia  [Viola caspia, an important ornamental and medicinal plant in northern and northwestern Iranian folklore, is distributed from Turkey in Europe and Crimea eastwards to northern Iran [V. reichenbachiana with seasonal cleistogamy was included as the biological background of the flowering process in Rostratae violets vs. V. caspia with the labile seasonal/simultaneous cleistogamy system.Both species studied herein, . Becker ,30. Violrn Iran) . Viola cern Iran ,33. The V. caspia with the implementation of additional research on constant floral traits allowing to distinguish CH and CL flowers and fruits at different stages of development in non-temporal cleistogamy. The main focus of the research was to test the influence of variable experimental conditions on the flowering timing of the CH/CL mating system in At the end of the growing season (starting from November/December) under short-day conditions, the meristems/buds of CH flowers were observed on the rhizome a–c. DormMeristems of CL flowers developed at CH flowering. To determine the location of CL meristems, the stem, petiole and CH peduncle anatomy was analyzed. All organs had characteristic wings; the vascular system was organized as a collateral bundle. In petioles, abaxial xylem and adaxial phloem formed an arched bundle e. In a pThe CL floral meristems were formed on elongated lateral branches in the upper leaf axils under long-day conditions in the angles between the lateral stem, the petiole and the peduncle of CH flowers h–m.V. reichenbachiana  with lines as the guide for pollinators, two lateral petals facing downwards and two upper petals slightly tilted  filled with seeds. At this stage, the erect style of the pistil ending with the stigma was still visible as a characteristic feature, allowing to distinguish the CH flower from the CL one with a curved style e,f. The semi-cleistogamous flowers of both species were smaller than the CH flowers and had all floral elements a–c, but V. reichenbachiana , followed by a long CL flowering ending in late November. At the end of CH flowering and at the beginning of CL flower development, SEMCL flowers  were produced . The CH flowering time lasted five months, starting in January and ending in May. Abundant CH flowering was observed in March and then the number of flowers per plant decreased. The CL flowering time was evidently shorter (three months), starting in March and ending in May, with the most CL flowers per plant in May . After MIn greenhouse conditions, CH and CL flowering duration was extended. CH flowering lasted six months (January-June) and CL flowering lasted twelve months (January-December). Abundant CL and CH flowering was observed in March . Both flp < 0.05). Such a correlation was not observed in the greenhouse conditions. In turn, in this temperature-stable conditions .These two habitats differed significantly in terms of mean daily temperature but much less so in the case of humidity . The chaV. caspia, but also to a gradual switch from seasonal cleistogamy (occurring in nature) to simultaneous CH and CL flower production. There were conspicuous differences in temperature between the Live Collection of Iranian Violets (LCIV) and greenhouse conditions. In LCIV, the daily temperature varied throughout the year ; in the greenhouse, it was more stable and did not drop below 20 °C. The daily temperature influenced the variable CH/CL ratio of V. caspia in LCIV and greenhouse conditions. The photoperiod, one of the main factors inducing CH/CL flowering in nature, was the same in the LCIV and greenhouse conditions , thus not influencing the differences in the CH/CL ratio in V. caspia.Changed environmental variables in this experimental study, modified the mixed CL/CH reproductive system. The greenhouse conditions, differing from natural conditions, led not only to a change in the flowering time of CH and CL flowers and the CH/CL ratio in Viola pubescens which clearly indicated that, in nature, seasonally variable environmental factors can drastically modify the mixed breeding system of this species [V. caspia, and indicate that the developmental program of cleistogamous species in nature might not be absolute/directional. The results from the greenhouse conditions clearly show that the daily temperature significantly affected the CH/CL flower ratio, with a clear predominance of CL flowers. This allows for long-term predictions of how global climate change (warming) will affect the CH/CL ratio of species with this reproductive system, which would have an impact on the reduction in population genetic differentiation.Our results add to the research on the impact of environmental variables on the development of the two flower morphs in cleistogamous species. Following the studies of  species , we showViola caspia is a very variable species in terms of both extreme phenotypic plasticity and habitat ecology (steppe to creek sides to deep beech forests) [Our results documented constant differences between CH and CL flowers at different stages of their development and at fruiting, allowing for the easy recognition of fruits derived from CH or CL flowers. CH flowers attract insect pollinators and promote cross-pollination and potential genetic diversity within populations ,38,39,40forests) ,42. Its forests) . The mosV. reichenbachiana and V. caspia in nature. In simultaneous cleistogamy, induced in V. caspia under greenhouse-controlled conditions, the flowering and fruiting of CH and CL occurred at the same time , and the distinction between CH and CL fruits was based on a constant pistil trait (curved style of CL pistil vs. straight in CH) which persists until fruiting. The traits of the CH and CL generative organs of V. reichenbachiana and V. caspia are not variable and not influenced by environmental conditions , similar to the stem and peduncle anatomy. Distinguishing CH and CL fruits of violets with seasonal cleistogamy in the field is not difficult because both flower types and fruits are temporally separated in the flowering season as in Modification of CH and CL bud fate by manipulation of environmental variables in experimental conditions opens a new area for further deep insight into the genetic regulation of chasmogamous/cleistogamous systems and could enable the control of outcrossing and self-fertilization in cleistogamous species.V. reichenbachiana, early dog violet, were grown in an artificial forest in Cracow . This species with seasonal cleistogamy served as a reference against V. caspia with the labile seasonal/simultaneous cleistogamy system.Specimens of Plants were observed over several seasons (2014–2017), and plant material was collected for determination of the location of floral meristems and flower structure.V. reichenbachiana specimens used in this study depended on the analysis: for CH flower meristem/buds location, ~10 plants were dug up, and the same plant number was used for CL meristem/buds location. For CH and CL flower morphology and microstructure, 4–5 flowers were collected from different plants, and 3–4 petioles or stem fragments were analyzed for organ anatomy.The number of V. caspia were randomly collected in their natural habitat in the Gilan-Saravan forest, located 13 km southwest of Sangar and 35 km from Rasht , and were transported to the Live Collection of Iranian Violets (LCIV) at the Research Institute of Modern Biological Techniques, University of Zanjan, Iran, located 150 km from Saravan forest .Forty specimens of v/v; Avantor Performance Materials Poland S.A., Gliwice, Poland) for 24 h and then stained with acetic orcein  and gently squashed to observe CH meristems. Chasmogamous floral meristems/buds were collected in December/January 2015/2016. The plants were gently dug from the soil to avoid damaging the root system and rhizome apices and inspected for CH buds using a stereoscopic microscope . Rhizome apices were fixed in a mixture of ethanol  and glacial acetic acid  for 48 h, gently squashed or cross-sectioned by hand.V. caspia has not been studied or documented because it is the same as that for V. reichenbachiana. Both species belong to the subsect. Rostratae of Viola section, in which floral features are conservative. The localization of CH and CL floral meristems/buds in All stages of CH and CL flowers of both species were analyzed during the season, and the differences in pistil shape, style and stamens between CH and CL flowers were documented with a stereoscopic microscope . Photographs were taken with an Olympus microscope  under 40× and 400× magnifications. V. reichenbachiana, CH and CL flowers were fixed in acetic acid–alcohol (FAA)  or in 4% glutaraldehyde  in 0.1 M Na cacodylate buffer , dehydrated in increasing concentrations of ethanol and dried at the critical point of carbon dioxide. Samples were glued onto holders, coated in gold  and examined in a Jeol JSM-5410 SEM  or in a Philips XL 30 SEM .For floral trait analyses of V. reichenbachiana stem, peduncle, petiole and CL flowers was analyzed on acetocarmine-stained handmade cross-sections as well as on microtome-made paraffin cross-sections stained with Heidenhain’s hematoxylin combined with Alcian blue according to the procedure described in [The anatomy of ribed in .Viola caspia stems and peduncles were fixed in FAA  for 48 h and then washed in 50% ethanol, dehydrated in a butanol series , embedded in paraffin wax , sectioned by rotary microtome . at a thickness of 20 μm, double s tained with crystal violet–erythrosine, cleared in xylene  and mounted in Canada balsam .http://www.zanjanmet.ir/, accessed on 27 August 2021).Twenty plants were cultivated in the LCIV and twenty plants were grown in pots (30 × 30 cm) containing garden soil—a mixture of sand, soil and rotten manure in equal proportions—in controlled greenhouse conditions. The photoperiod was supplied naturally according to the conditions of the season. A gradual increase in temperature and in the photoperiod from late winter to early spring as well as gradual cooling in autumn were more evident in nature than in the greenhouse, especially the gradual increase in temperature . The datObservation of CH and CL flowering and fruiting was carried out on plants growing in the greenhouse and in the LCIV throughout the season for five years (2015–2020). The number of CH and CL flowers per plant was counted in each month of the season. Every day, the open CH flowers and CL flowers were marked with a red label to avoid counting the same flowers in the following days.The correlations  between four parameters  in both conditions (greenhouse and LCIV) were counted in the stats package in R v. 3.6.3 .(1) Environmental variables influence the timing of CH and CL flowering and the CH/CL ratio. The controlled greenhouse conditions may allow manipulation of this mixed mating system and even induce simultaneous flowering of CH and CL flowers in species with temporal separation of these two flower morphs in nature.(2) Determination of the location of floral meristems is important for further molecular research on the differential expression of genes involved in the development of CL and CH flowers.Viola.(3) The differences in fruit morphology between cross-pollinated CH and obligate self-pollinated CL flowers are important in population genetic diversity and in breeding programs of


This study aimed to identify people at risk of cardiovascular diseases (CVD) using the Framingham risk score (FRS) and to examine their willingness to modify their lifestyle. A descriptive correlational study with 1229 participants, aged 30–74 years, without cardiovascular or cerebrovascular diseases who visited a health examination center in South Korea was conducted. Of 1229 participants, 455 were identified as high risk for CVD with an FRS of 10 or higher. A logistic regression analysis confirmed age, sex, muscle training, and weekly drinking frequency as predictors of intention to quit smoking; age, smoking, and waist to height ratio (WHtR) as predictors of intention to quit drinking; sex and WHtR as predictors of intention to engage in physical exercise; and hypertension and body mass index as predictors of intention of muscle training. People at high risk for CVD lack willingness to modify their lifestyle, and aggressive, customized intervention is needed to promote lifestyle modification. Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the 2019 Statistics Korea report, it is the second leading cause of death following malignant neoplasm, with 117.4 deaths per 100,000 people [In 2014, Korea designated the first week of every September as the cardiovascular and cerebrovascular diseases prevention week, during which the government strives to reduce CVD mortality by launching the Red Circle Campaign. It increases awareness of and promotes compliance with the prevention and management of severe cardiovascular diseases such as myocardial infarction and stroke .Considering that heart disease remains one of the leading causes of death despite advances in medical technology and development of novel drugs, it is important to note that other factors contribute to the incidence of heart disease. While many countries have strived to regulate these CVD risk factors and have announced their success in reducing CVD mortality , the KorCardiovascular disease risk factors can be categorized into two groups: modifiable and non-modifiable risk factors. Non-modifiable cardiovascular disease risk factors are those that cannot be changed. These include a person’s age, ethnicity, and family history (genetics cannot be changed), among other factors. Modifiable cardiovascular disease risk factors are those that can be reduced or controlled with behavioral changes .Some of the preventable risk factors of CVD include smoking, hypertension, DM, hyperlipidemia, obesity, and lack of exercise ,8,9. FurThe Framingham risk score (FRS) is a simple tool widely used to predict the level of CVD risk in the next 10 years, and it provides guidelines for managing risk factors ,12. In pA Korean study using FRS confirmed the factors affecting FRS by sex , and ano-Among participants with moderate or high cardiovascular risk, the better the lifestyle habits, the higher their intention to quit smoking.-Among participants with moderate or high cardiovascular risk, the better the lifestyle habits, the higher their intention to stop drinking.-Among participants with moderate or high cardiovascular risk, the better the lifestyle habits, the higher their intention to engage in physical activity.-Among participants with moderate or high cardiovascular risk, the better the lifestyle habits, the higher their intention to engage in strength exercises.The purpose of the present study was to classify risk groups using FRS and to identify the factors that influence the willingness to improve lifestyle, which is a modifiable risk factor. We hypothesized the following:This study employed a cross-sectional study design to identify, through influencing with CVD risk, participants’ intentions to modify their lifestyle habits.A total of 1282 people aged 30–74 years  who undeParticipants’ demographic and lifestyle-related information, namely, age, sex, drinking, and smoking, was surveyed using the pre-exam health questionnaire. Information about physical activity was collected using the International Physical Activity Questionnaire (IPAQ) , specifiFor the blood test, the participants were informed in advance to refrain from overeating, drinking, and smoking, and to skip their medications as they may affect the test results. All participants fasted for at least eight hours prior to the blood test. Fasting glucose, HDL-C, total cholesterol, aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and creatinine (Cr) levels were analyzed. Blood tests were performed by three clinical pathologists with more than 10 years of experience working at the institution where this study was conducted. The pathologists receive a training session once a year on drawing blood accurately.Participants’ intentions to improve their lifestyle pertaining to smoking, drinking, physical exercise, and muscle training were examined using a self-report questionnaire, with “yes” or “no” responses.The FRS was calculated by scoring participants’ age, HDL-C, total cholesterol, SBP, DM, and smoking status according to their sex, as per the calculation method presented in the Framingham heart study . This caThis study was approved by the Institutional Review Board of the research facility (IRB No. 130750-202002-HR-001) prior to data collection. After explaining the purpose of this study to the research participants, written informed consent was obtained from the participants prior to the health examination.The data were analyzed using SPSS/WIN 23.0 software . The general characteristics were analyzed using descriptive statistics, namely, frequency and percentage. Differences in demographics and willingness to modify lifestyle according to the CVD risk category were analyzed with a one-way analysis of variance (ANOVA). The predictors of willingness to modify lifestyle in 455 participants in the intermediate-risk and high-risk groups (FRS ≥ 10%) were analyzed using binary logistic analysis.F = 135.1, p < 0.001). In terms of sex, 667 (87.5%) in the low-risk group and 149 (60.6%) in the intermediate-risk group were women, while 184 (88.0%) in the high-risk group were men.The mean age significantly differed among the low-risk group (54.6 ± 10.7 years), intermediate-risk group (62.0 ± 7.7 years), and high-risk group (65.4 ± 5.7 years) , and high-risk group  . The percentage of current smokers also significantly differed in the high-risk group  compared to the low-risk and intermediate-risk groups . The percentage of people who drank alcohol at least once a week was significantly higher in the high-risk group  . In terms of intention to quit smoking, the majority in all three groups had no intention to quit smoking, with a significant difference among the groups . In terms of intention to quit drinking, only 28.2% of the high-risk group were willing to quit drinking, with a significant difference among the three groups  , intermediate-risk group (= 0.012) .2), with a significant difference among the three groups . Waist to height ratio (WHtR) was high in the intermediate- and high-risk groups (0.53 ± 0.1), with a significant difference among the three groups .The predictors of intention to modify lifestyle in at-risk CVD groups were analyzed for 455 participants in the intermediate-risk (FRS ≥ 10%) and high-risk groups (FRS ≥ 19%) .2 = 294.09, p < 0.001), with 41.8% of the variance explained , with 24.3% of the variance explained , with 7% of the variance explained , with 3.9% of the variance explained. Intention to engage in muscle training was 1.85-times higher in those with hypertension than in those who were not diagnosed with hypertension  and 1.69-times higher among those with a BMI > 25 kg/m2 than in those with a lower BMI  (The model for identifying the predictors of intention to engage in muscle training in the at-risk CVD groups was significant (χ23–2.32) .This study presents valuable foundational data for exploring fundamental strategies to lower the incidence of cardiovascular and cerebrovascular disease and develop intervention programs that improve compliance by identifying people’s willingness to modify their lifestyle according to their risk of cardiovascular and cerebrovascular disease. This study may provide the basis for planning and implementing health measures aimed at reducing modifiable risk factors for cardiovascular diseases .In this study, intention to quit smoking was higher with increasing age in women than in men, with higher muscle training frequency, and with higher weekly drinking frequency in people at an intermediate or a high risk for cardiovascular and cerebrovascular disease (FRS ≥ 10%). These results are contradictory to previous findings on adults and older adults in public health centers in Korea, where men were more willing to quit smoking than women ,22. A prThe higher intention to quit smoking among women in our study seems to reflect the social trend that still renders female smoking taboo. Furthermore, people who frequently engaged in muscle training were more willing to quit smoking, suggesting that people who practice health behaviors are more likely to self-examine the effects of their health behaviors and modify their lifestyle . Thus, sIn this study, old age, elevated WHtR, and current smoking were identified as the predictors of intention to quit drinking. Similarly, a previous cohort study on adults aged 40–69 years showed that tendency to quit drinking increased with advancing age . FurtherIncreased WHtR indicates abdominal obesity and because the risk for CVD is higher in those with greater visceral fat even with the same body weight , managemIn the present study, hypertension and obesity were confirmed as the predictors of intention to engage in muscle training in at-risk CVD groups. As emphasized by the AHA guidelines , with thAdditionally, in terms of lifestyle and comorbidities according to FRS in this study, the rates of smoking and drinking in the high-risk group were 75.6% and 41.6%, respectively, while the percentages of those engaging in high-intensity exercise and muscle training were 12.4% and 24.9%, respectively. This shows that people at high risk for CVD fail to modify their lifestyle even though they need to further strive to reduce their risk factors and manage their health .This study has a few limitations. First, the sample was conveniently sampled, thus the findings have limited generalizability. Therefore, a multicenter study that utilizes tools to examine various cardiovascular and cerebrovascular disease risk factors should be conducted. Second, this was a cross-sectional study that investigated the intention to modify lifestyle using a self-report questionnaire, so respondent bias may exist. In addition, causality between cardiovascular disease and lifestyle cannot be determined. Therefore, longitudinal studies that implement a long-term lifestyle modification intervention and examine its effects on reducing the prevalence of cardiovascular and cerebrovascular disease are needed. Despite these limitations, this study is meaningful in that it confirmed the intention to modify the lifestyle habits for the risk factors of cardiovascular disease, centering on those with high cardiovascular risk who actually need lifestyle improvement.This study identified the predictors of intention to modify lifestyle behaviors like smoking, drinking, physical activity, and muscle training, in people at risk for CVD. The findings were based on the FRS data of 1229 individuals aged 30–74 years without cardiovascular and cerebrovascular disease who visited a health examination center in Korea.The results showed that old age, being female, high muscle training frequency, and high drinking frequency are predictors of intention to quit smoking, while old age, low WHtR, and current smoking are the predictors of intention to quit drinking in at-risk CVD groups. Further, intention to engage in physical activity was 0.27-times lower among women; that is, intention to engage in physical activity was higher for males and those with low WHtR , whereas the intention to engage in muscle training was higher in those with hypertension and higher BMI.Intention needs to be supplemented by other, more proximal factors that might compromise or facilitate the enactment of intentions. These differences can be overcome by promoting self-efficacy for behavior modification and establishing educational strategies for specific behavior modification, which may help bridge the intention–behavior gap.


Catha edulis Forsk) is a plant consumed by many people in Eastern Africa, including Ethiopia, and Southern Arabia to be stimulated. There are several human and animal studies on khat that provide information about its toxic effects. However, the potential toxic effects of khat on embryos and fetuses have not been elucidated. The aim of the present study was to investigate the embryotoxic and fetotoxic effects of khat exposure during the earliest period of gestation in rats. Pregnant Wistar albino rats were treated with khat extract at 250, 500, and 750 mg/kg doses from day 6 through day 12 of gestation. The treatment was delivered by gavage. Embryos and fetuses were recovered on gestational day 12 or day 20, respectively, and were quantitatively and qualitatively assessed for developmental anomalies. Placentae from the treatment and control groups were investigated for histopathological effects. Results of the present study showed that khat exposure during pregnancy had dose-dependent toxic effects in rat embryos and fetuses. Prenatal growth retardation such as reduced fetal weight and crown-rump length was observed in near-term fetuses, especially, in animals treated with the highest dose of khat (p < 0.05). Growth retardation and developmental anomalies were also observed in day 12 embryos of khat-treated rats. Maternal weight gain of the khat-treated group was also significantly lower than the control group. Cytolysis, decidual hypoplasia, and atrophy were observed in the placenta of the khat-treated rats. Findings of the present study revealed, for the first time, that exposure of pregnant rat to crude extract of khat causes embryotoxic and fetotoxic effects.Khat ( Catha edulis Forsk) is extensive in Ethiopia. It is also widely consumed in eastern Africa, including the Horn of Africa [The consumption of khat . By this, it is mainly used as a recreational drug which helps to attain a state of euphoria and stimulation . Khat usBased on recent research reports, consuming khat is now a worldwide phenomenon; users are now found in Europe and North America as well . Khat usEarlier study report indicates that the prevalence of khat chewing was higher in male compared with female, whereas, recent reports, in some countries, revealed sharp escalation in khat use by females. In Ethiopia, recent estimates of khat chewing prevalence reach up to 50%, with 17% self-described as daily users, predominantly men  . Recent Based on a study conducted in Yemen, on 1141 pregnant women, the prevalence of low-birth-weight babies was higher in khat chewer mothers compared with nonchewers . HoweverAlthough there are data on different adverse effects of khat, very little is known about its effects on the embryos/fetuses. Therefore, the main purpose of this study is to evaluate the embryotoxicity and fetotoxicity of khat in rats.ad libitum. Pregnant rats were weighed on the 1st, 6th, 12th, and 20th day of pregnancy (for day 20 experiment) and on the 1st, 6th, and 12th day of gestation (for day 12 experiment).The experiments are designed to study the embryotoxicity/fetotoxicity of khat in Wistar rats. Experiments were carried out in day 20 fetuses (day 20 experiment) and in day 12 embryos (day 12 experiment). We followed the methods of Seyoum and Persaud , Seyoum Fresh leaves of khat (about 7000 g) were purchased at a local market in Aweday, 515 km east of Addis Ababa, Ethiopia, where it is commonly grown. The fresh bundles were packed in plastic bags and transported in an icebox to the laboratory. The fresh leaves were immediately kept in a deep freezer (-20°C). Plant identification, by a taxonomist, was carried out at the National Herbarium, College of Natural and Computational Sciences, Addis Ababa University, Ethiopia, and a voucher specimen (collection number SB-001) was deposited for future reference.The leaves were chopped with a knife in a dark place, weighed by electronic digital balance, and put into an Erlenmeyer flask containing 80% methanol. Enough volume of solvent was added so as it covers the crushed plant material in the flask; then, the flask was sealed with aluminum foil and frequently agitated using a rotary shaker at 120 rpm for 24 h. It was then filtered using a Whatman No. 1 filter paper  and macerated 3x for a total of 72 h and filtered. The combined filtrates were concentrated using a rotary evaporator  at a temperature not exceeding 40°C. The concentrated extract was then transferred into a freeze-drying flask and lyophilized . The dried extract was kept in a tightly sealed container at -20°C until use.Nulliparous female albino Wistar rats weighing 200 to 230 g and sexually matured males of proven fertility were used. The rats were obtained from the animal breeding house of the Ethiopian Public Health Institute (EPHI). The rats were accommodated in stainless-steel cages where the temperature (22-23°C) and relative humidity (50% ± 10) of the room were controlled and 12 h of light and 12 h of dark were maintained. During the period of adaptation, all the animals received food (Wayne F6 Rodent Blox Pellets) and tap water. The handling of animals and all experimental procedures were conducted in compliance with the internationally accepted Organization for Economic Co-operation and Development (OECD) guideline , and thead libitum control group and received the pellet diet and water unrestricted. Each animal in the experimental groups  was given the respective doses of khat and the pellet diet and water ad libitum. The pair-fed control group animals were restricted-fed and received an equal amount of the same pellet diet and water only. The treatment period was from day 6 to day 12 of gestation, since this period is an active period of organ formation in rats [In both day 12 and day 20 experiments, a total of 50 pregnant rats were randomly divided into five groups. In each experiment, five rats were assigned to each group. Group I received vehicle and served as the pair-fed control group (CON). Groups II, III, and IV were treated with three doses of the crude khat extract: 250 mg/kg (K250), 500 mg/kg (K500), and 750 mg/kg (K750), respectively. Group V served as the untouched  in rats .The three treatment doses of khat extract were chosen based on previous study reports. The khat extract was weighed and mixed with 1.0 ml of the vehicle, and gavage was used for oral administration. Such route of administration was selected since the khat leaves are usually consumed orally. In addition, pharmacokinetic studies have shown that khat is readily absorbed into the blood from the gastrointestinal system .in vivo investigation [This experiment was planned to investigate possible embryotoxic effects of khat on rat embryos. On day 12 of gestation, at 12:00 hours, the animals were anesthetized with an intraperitoneal injection of pentobarbital. The gravid uterine horns were taken out of the abdomen and placed in a Petri dish containing normal saline. Each horn of the uterus was incised along the antimesometrial border to disclose the embryos covered with membranes. The surrounding embryonic membranes were curiously removed by using fine forceps. Thereupon, the visceral yolk sac was revealed and yolk sac circulation was examined. Once the embryonic membranes were removed, the embryos were detached from the corresponding placenta. Then, different body systems of the embryos were evaluated for developmental delays, namely, circulatory, nervous, visual, auditory, olfactory, and skeletal systems as well as the craniofacial region. All embryos were evaluated for developmental delays according to the Brown and Fabro morphological scoring system , which wtigation . In addiThis experiment was designed to study gross congenital anomalies (fetotoxic effects) caused by exposure to khat in pregnant rats. On gestational day 20, gravid females were anesthetized with an intraperitoneal injection of pentobarbital; the uterine horns were exposed and examined intact. The number of implantation sites  and resorptions  was counted. Live or dead fetuses were determined by applying a gentle pressure on them. The fetuses, covered with membranes and attached with placenta, were disclosed by incising the uterine horns along the antimesometrial border. Then, each fetus was detached from the corresponding placenta, and its weight as well as crown-rump length (CRL) was measured. The weight of the placenta also was recorded. Following these measurements, all fetuses were examined for gross external malformations.Gross external examination was conducted to check for possible treatment-related external malformations in the limbs, vertebral column, tail, external genitalia, and craniofacial regions of the fetuses.μm thickness using a Sorvall-JB microtome. The sections were then placed on glass slides, dewaxed, dehydrated, and stained with hematoxylin and eosin (H&E). Finally, the stained slides were covered with a coverslip for microscopic examination. The sections were examined for evidence of structural and vascular alterations using a Nikon binocular light microscope. Decidual zone, labyrinthine zone, giant cells, and trophoblasts of the placenta were examined and used as indices of functional as well as structural changes in the placenta.Three to four placentae from each group were randomly selected and put in 10% formalin. The fixed placentae were transferred into 70% ethyl alcohol overnight. Following routine processing for light microscopy, the placentae were blocked in paraffin and cut into sections of 4 Stained tissue sections of the placenta were carefully examined by a pathologist under a binocular compound light microscope. Tissue sections from the experimental groups were examined for any evidence of histopathological changes with respect to those of the controls. After examination, representative photomicrographs were taken by using an automated built-in digital photo camera  under a magnification of ×20 objective.The data, concerning weight gain, pregnancy outcomes, maternal food intake, fetal growth, and developmental anomalies in each group, were analyzed by one-way analysis of variance (ANOVA) using the SPSS version 20. The data regarding embryonic development and histopathological analysis was analyzed by using chi-square analysis.Treatment of pregnant rats with khat at doses of 250 mg/kg, 500 mg/kg, and 750 mg/kg body weight, from days 6 to 12 of gestation produced a dose-dependent decrease in body weight gain of the dams. The decrease in the body weight gain was significant in all khat-treated groups compared to that in the control group .ad libitum control group. The K750 showed a significantly higher incidence of fetal resorptions and also a significant decrease in the number of implantation sites compared to the pair-fed control and ad libitum groups (p < 0.05)  .p < 0.05) in embryos of K500 and K750 groups   Tables .p < 0.05) compared to the pair-fed control and ad libitum groups. However, the development of mandibular process was not affected , and the number of fetuses also was significantly decreased (p < 0.05) in the K750 mg/kg group compared to the pair-fed control and ad libitum groups. Implantation sites and the number of live fetuses per litter were significantly reduced at the dose of 750 mg/kg compared to that in both the pair-fed control and the unrestricted-fed ad libitum groups (p < 0.05)  . The numad libitum control groups. Both CRL and fetal weight showed a significant decrease in the high-dose treated group (K750)  of khat extract compared with pair-fed and p (K750) . No grosp (K750) .ad libitum and pair-fed control groups. Dose-dependent pathological changes in the placenta were observed. These microscopic structural changes were seen in the decidua basalis, trophoblastic zone, and labyrinth zone , with less branching in the treatment group at the highest dose Figures . The obsDecidua basalis, thin layer at the base of the placenta, is an important site for maternal angiogenesis. The decidua basalis includes newly developed blood vessels, which play essential roles in the development of vascularized decidual-placental interface. The decidua can produce a wide range of hormones, cytokines, growth factors, and immunomodulatory molecules involved in the recruitment of the limited but specific immune cell populations and growth of the placenta . FindingIn the present investigation, the embryotoxic/fetotoxic and toxic effects of khat were evidenced by a significant delay in embryonic and fetal development and decreased maternal weight gain during the period of gestation. This study, for the first time, has also demonstrated that khat has pathological effects on histological features of rat placenta at a cellular level. Based on the presented data, it can be concluded that administration of crude extract of khat to pregnant rats may not be safe. Since embryotoxic/fetotoxic effects of khat have now been established experimentally, its use, especially during pregnancy, should be strongly discouraged.


This cross-sectional study was performed on patients with pemphigus vulgaris referred to Faghihi Hospital and Shiraz Dental Faculty in 2017-2018. The participants included 26 women with histopathologically confirmed pemphigus vulgaris and 26 healthy age-matched controls. The serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen, progesterone, testosterone, prolactin, dehydroepiandrosterone (DHEA), and dihydrotestosterone (DHT) were evaluated in both groups. Independent p = 0.015 and p = 0.026, respectively).  The mean age of the patients was 49.88 ± 10.46 years and that of the control group was 49.92 ± 11.30 years. Unlike the case group, the DHEA serum level was significantly higher among nonmenopausal participants in the control group. Moreover, the levels of testosterone and DHEA were significantly lower in the case group in comparison to the control group ( Considering the effects of age and menopause, the serum levels of testosterone and DHEA were significantly lower in the patients with pemphigus vulgaris than in the healthy controls. Hence, these hormones might have a role in the pathogenesis of pemphigus vulgaris. Pemphigus vulgaris is a common autoimmune blistering disease, i.e., the most common form of pemphigus, with the incidence of 2-10/1,000,000 . In thisGenerally, there is a clear sex tendency in the prevalence of most autoimmune diseases, with females being affected more . AdditioHormonal changes in menopause cause general manifestations, which are concerning due to the increase in life expectancy. Decreased levels of progesterone, especially estrogen, and secondary alterations in FSH and LH are responsible for clinical manifestations postmenopause. In addition to the general manifestations of menopause such as hot flushes and psychological alterations, oral symptoms including burning mouth syndrome and dry mouth (xerostomia), some other disorders such as lichen planus, benign pemphigoid, and Sjogren's syndrome, and a rise in the prevalence of periodontal diseases have been reported in few studies . There aUp to now, several studies have been conducted on the relationship between sex hormones and induction of flares as well as treatment or even incidence of autoimmune diseases. For instance, some studies have indicated an association between menopause and age at the peak of rheumatoid arthritis incidence . BesidesThis cross-sectional study was performed on patients with pemphigus vulgaris referred to Faghihi Hospital and Oral and Maxillofacial Disease Department of Shiraz Dental Faculty in 2017-2018.The study protocol was concordant with the ethical principles of Helsinki (version 2002) and was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.DENTAL.REC.1399.136).Patients with histopathologically confirmed pemphigus vulgaris who were admitted or treated in the outpatient clinic were enrolled into the case group, and healthy age-matched women were considered as the control group. Since there was no similar study for sample size assessment, the results of the most similar study on an autoimmune disease was taken into account. Considering the power of 80%, type 1 error of 0.05, and correlation coefficient of 0.58, a 21-subject sample size was estimated for each group. Considering a 10% dropout, 26 participants were enrolled into each study group . Prior tThe serum levels of sex hormones were assessed. In doing so, blood samples were obtained during routine evaluation before initiating corticosteroid therapy two hours after waking up on day three of the menstrual cycle. It is noteworthy that it was not necessary to specify a day for obtaining blood samples from menopausal women. The serum levels of LH, FSH, estrogen, progesterone, testosterone, prolactin, DHEA, and dihydrotestosterone (DHT) were evaluated via ELISA (monobind) Padtan Elm (EIA) kits  in Motahari laboratory. The participants' demographic data including age, disease extension, and menopause status were recorded, as well.t-test was utilized to compare the case and control groups regarding the mean levels of hormones. In addition, two-way ANOVA was used to compare the two study groups regarding the mean levels of hormones by taking the effect of age and menopause status into account. p < 0.05 was considered statistically significant.The data were analyzed using the SPSS software, version 18. Independent This study is aimed at evaluating the serum levels of sex hormones in 26 females with pemphigus vulgaris and 26 age-matched healthy controls. The mean age of the patients was 49.88 ± 10.46 years and that of the control group was 49.92 ± 11.30 years.p = 0.015 and p = 0.026, respectively).The mean serum levels of all the evaluated hormones have been presented in p = 0.015 and p = 0.026, respectively).Considering the effect of age on the serum levels of sex hormones based on the results of two-way ANOVA, the difference between the two groups regarding sex hormones' serum levels have been presented in p = 0.001, p = 0.019, and p = 0.044, respectively).The mean serum levels of sex hormones among menopausal and nonmenopausal participants in each study group have been shown in p = 0.017 and p = 0.047, respectively).Considering the effect of menopause status on the serum levels of sex hormones based on the results of two-way ANOVA, the difference between the menopausal and nonmenopausal participants was evaluated in each study group . Accordip = 0.003)  .p value for estrogen was not statistically significant, it was considerable in both the effect of menopause (p = 0.061) and the effect of age (p = 0.054) on the serum levels of sex hormones. In case a larger number of participants were enrolled into the case group, significant results might have been obtained.In this study, the serum levels of FSH and LH were significantly higher in menopausal participants than in nonmenopausal ones. However, the serum level of estrogen was significantly higher in nonmenopausal participants in both study groups. These differences were concordant with the effect of age on the natural changes in the serum levels of these hormones. In the control group, the DHEA serum level was significantly higher in nonmenopausal participants, while this was not the case among the patients with pemphigus vulgaris, which might indicate some hormonal disturbances in these patients. Considering the effect of age on the serum levels of sex hormones, significantly lower levels of DHEA and testosterone were observed in the patients with pemphigus vulgaris compared to the healthy controls. In this comparison, although the The previous studies assessed the role of sex hormones in immune system regulation, activation, or suppression. Accordingly, there are several theories on the effects of these hormones on autoimmune situations. However, these theories are sometimes controversial. Considering pemphigus vulgaris as an autoimmune disease, researchers have made attempts to evaluate the role of sexual hormones in the pathogenesis of this disease. To the best of the authors' knowledge, no similar study has been conducted on pemphigus vulgaris. Nonetheless, other autoimmune diseases have been evaluated in both human and animal models using different methods and concepts. For instance, Forsblad-d'Elia et al. evaluated the effect of daily consumption of 50 mg oral DHEA in primary Sjogren among postmenopausal women. They reported the relief of the dry mouth symptom as well as an increase in the levels of testosteron, estrogen, and DHEA in the case group in comparison to the placebo group . These rp = 0.026) and testostrone (p = 0.015) were significantly lower in the patients affected by pemphigus than in the healthy controls. This was consistent with the results of similar studies on Sjogren's syndrome [The current study findings revealed that the serum levels of DHEA (syndrome –13, thyrsyndrome , rheumate p = 0.0 were sigsyndrome , and syssyndrome , which ssyndrome .There are some reports about the possible interrelation between DHEA and soluble immune mediators, which can affect the function and adhesion of leukocytes in females with SLE . In anotStudies on sex hormones are not limited to androgens and testosterone. A prior study indicated no significant relationships between exposure to estrogen or prolactin and increased risk of lupus. That study as well as other studies assumed that early menopause could be associated with an increased risk of SLE development , 25. MorA previous study revealed an immunosuppressant activity, especially delayed type of hypersensitivity, for estriol. This hormone therapy was suggested for treating multiple sclerosis . AlthougThe adrenal glands in humans secrete DHEA, especially DHEA-S, which can transform to androgen or estrogen in peripheral tissues . Thus, tTestosterone can be secreted by the ovaries, adrenal glands, and peripheral tissues. The proportion of testosterone secretion from these sources can vary by aging. In fact, the serum level of this hormone is affected by aging rather than by menopause. Thus, a woman in her forth decade of life has half of the testosterone level as a woman in her second decade of life , 34. TheMenopause exerts a negative impact on women's health. This situation that is associated with a progressive decrease in sex steroids followed by estrogen secretion arrest can be related to many morbidities. In fact, a marked decline in estradiol and DHEA after menopause is a critical challenge among females. The present study results indicated no significant reduction in the estrogen serum level among the patients with pemphigus vulgaris. Yet, choosing a larger sample size might have led to more precise results.The present study had some limitations related to the small sample size and patient selection. Considering a healthy control group, using a larger sample size, evaluation of all effective factors in hormone levels could lead to more reliable results. Furthermore, a controlled study is recommended to be performed on the effect of hormone therapy using estrogen in an animal in order to explore the risk of endometrial cancer. Hormone therapy or balancing sex hormones, especially DHEA and testosterone, that were more significant in this study can provide a new insight for controlling, preventing, or even treating pemphigus vulgaris, which should be evaluated precisely in future studies.Considering the effects of age and menopause status, the serum levels of testosterone and DHEA were significantly lower in the patients with pemphigus vulgaris than in the healthy participants. Therefore, these hormones may play a role in the pathogenesis of pemphigus vulgaris.


The purpose of this study was to explore the structural and functional asymmetry of precentral and postcentral gyrus in patients with unilateral chronic shoulder pain (CSP) utilizing MRI.We collected structural and resting-state functional MRI (rs-fMRI) data in 22 left-sided, 15 patients with right-sided CSP, and 24 healthy controls (HCs). Here, we performed the structural asymmetry and seed-based functional connectivity (FC) analyses. We extracted regional cortical thickness and surface area measurements from T1-weighted MRI images, using asymmetry indexes (AIs) to assess asymmetries. We used Data Processing and Analysis for Brain Imaging software for seed-based FC analysis and selected unilateral-precentral and postcentral as the regions of interest. Then, we performed group comparisons of the neuroimaging metrics, and also explored the relationships between brain asymmetry and clinical variables.We found significant differences in surface area AIs of the precentral among three groups, the AI values were negatively correlated with the visual analog scale score and positively correlated with Constant–Murley scores (CMS) in the left-sided CSP group. Further, FC of left postcentral with cingulate gyrus and left paracentral lobule showed significant group differences; FC of right postcentral with left caudate, left paracentral, and left postcentral were different among groups; FC of right precentral with the cingulate gyrus, precuneus, and left paracentral revealed significant group differences. Besides, there was a positive correlation between right precentral-cingulate gyrus FC and CMS in the right-sided CSP group.Surface area and FC patterns asymmetry exist in precentral and postcentral gyrus in patients with unilateral CSP. Asymmetry trend is associated with pain severity and shoulder joint function impairment. Brain structural and functional asymmetry may be an important indicator for understanding the potential mechanism of chronic pain. In a variety of musculoskeletal disorders, chronic shoulder pain (CSP) is ranked third for the number of patients . EpidemiAccording to the concept of homunculus , the corThe asymmetry of the structure and function of the two hemispheres is a distinctive feature of human brain . HemisphHere, this study focuses on assessing the structure and function asymmetry in precentral and postcentral regions in patients with unilateral CSP. We used the asymmetry index (AI) , 18 to aIn this study, 39 patients with unilateral CSP were recruited between December 2016 and July 2017. Moreover, we recruited 26 healthy controls, matching the age and gender of our patients (from March 2017 to December 2018). More details about the recruitment of the CSP and HC participants  were desThis study was conducted in agreement with the Declaration of Helsinki. We collected MRI data from all participants. Before MRI scanning, VAS score –22 and CMRI images were obtained at a Siemens 3.0 T MRI scanner  using a standard head coil at the Department of Radiology for Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University. The high-resolution T1 MRI was acquired using gradient echo sequence with the following parameters: repetition time (TR) = 2,300 ms, echo time (TE) = 2.32 ms, flip angle (FA) = 8°, inversion time = 900 ms, field of view (FOV) = 240 × 240 mm, number of slices = 192, voxel size = 0.9375 × 0.9375 × 0.9 mm, and in-plane resolution = 256 × 256. In addition, the resting-state functional MRI (rs-fMRI) was scanned using echo planar imaging (EPI) sequence: TR = 2,000 ms, TE = 30 ms, FOV = 220 × 220 mm, FA = 90°, slice thickness/gap = 3.5/0.6 mm, axial slices = 33, in-plane resolution= 64 × 64, and 240 volumes. We used comfortable foam pads to minimize the head motion and earplugs to reduce noise interference. Before starting the scanning, we instructed participants to keep their eyes closed, stay awake, avoid engaging in any specific thoughts, and keep still.http://surfer.nmr.mgh.harvard.edu/, V6.0) to process the structural MRI data to reconstruct the cortical surface. Mean cortical thickness and surface area were derived for each of the 68 cortical regions of the Desikan-Killiany Atlas (34 per hemisphere). Cortical thickness was estimated for each participant using the distance from the white matter boundary to the corresponding pial surface   data were processed using the software MATLAB 2013b  and the toolkit of Data Processing and Analysis for Brain Imaging  . For eacp = 0.126).Four participants (two patients and 2 HCs) were excluded from the study on account of excessive head motion (>3 mm in translation or >3.0° in rotation) during the rs-fMRI scanning. As a result, 22 patients with LCSP, 15 patients with RCSP, and 24 HCs were included in further statistical analyses. Furthermore, we extracted the mean framewise displacement (FD) for each participant to measure the extent of head motion , 27 and t-test for CMS, and Mann–Whitney U-non-parametric tests for VAS and pain duration. As for categorical variables , we used the chi-square test to evaluate the differences among groups.We used SPSS statistics 21 software to conduct statistical analyses. Before statistical analyses, we checked the normality of each metric. Age and CMS were normally distributed in each group, whereas pain degree (VAS score) and pain duration were non-normally distributed. We used one-way ANOVA for age, two-sample To compare the difference in cortex structural asymmetry of precentral and postcentral regions related to unilateral CSP, AI for each cortical metric is calculated as a widely used formula :post-hoc analysis.Consequently, a negative AI reflects a rightward asymmetry and a positive AI means that asymmetry is shifted to the left. In the three groups, we used ANOVA or non-parametric tests to compare the difference in asymmetrical changes in cortical surface area and cortical thickness, and then performed the r-to-z transformation was used to convert each final FC map of individual to z-value maps. Among three groups, we used one-way ANOVA analyses to obtain the F statistical map. Finally, Gaussian Random Field (GRF) theory multiple comparison correction (p < 0.01 and cluster-level p < 0.05).For the functional analyses, we selected left precentral, right precentral, left postcentral, and right postcentral from the Automated Anatomical Labeling (AAL) atlas  as the srrection  was usedp < 0.05).We extracted FC value between the seed and the region with significant group differences. For normally distributed variables, Pearson's correlation was used to analyze the correlation between AI and FC values and VAS and CMS. For non-normally-distributed variables, we used Spearman's correlation analysis. The above statistical analysis used SPSS software . The pain duration , VAS , and CMS  showed no significant differences in patients with LCSP and RCSP. More details are presented in Among the three groups, there was no significant difference here in demographic data . According to the post-hoc pairwise comparison analyses, there was a significant difference in AI between the LCSP and RCSP groups , specifically, the LCSP group showed rightward asymmetries (group means AI = −0.017), and the RCSP group showed leftward asymmetries (group means AI = 0.043) in the precentral surface area  and a negative correlation with the VAS scores  in the patients with LCSP  in the RCSP group  in patients with LCSP patients . In addition, we found these structural and functional asymmetry metrics were correlated with clinical parameters. Specifically, AI values of precentral in LCSP had a significant positive correlation with CMS scores and a negative correlation with the VAS scores. Right precentral—cingulate gyrus FC had a significant positive correlation with CMS scores in RCSP. Our findings may clarify the neural mechanism of CSP from the perspective of brain asymmetry.post-hoc difference was observed only between RCSP and LCSP groups and not between HC and patient groups. Here, the AI of the precentral area had a tendency of rightward asymmetry in the LCSP group, and a tendency of leftward in the RCSP group. The AI of HC was in between. These findings indicate that the precentral areas of the contralateral and ipsilateral to the shoulder pain side are affected asymmetrically. This is consistent with the left-right cross characteristic of sensory/motional projection that the precentral may be more involved to the pain and function of the contralateral shoulder. Our results may further support the lateralization of the brain in pain processing , and altered functioning between networks in patients.In summary, our study found that there was significant asymmetry in the cortical surface area of the precentral in patients with CSP, and the asymmetry value of patients with LCSP has a clear correlation with the severity of the condition of patient. Additionally, the precentral and postcentral gyrus in different hemispheres had different FC patterns with the whole brain regions in patients with CSP. What's more, the FC between the right precentral and cingulate gyrus was correlated with the condition of patient in the RCSP group. Our research adds to the literature suggesting a critical role of precentral and postcentral in the pathophysiology of CSP, and the brain asymmetry effect may be an important hallmark of chronic pain diseases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of the Beijing Hospital of Traditional Chinese Medicine, affiliated with Capital Medical University. The patients/participants provided their written informed consent to participate in this study.XW designed this work. XYW, HZ, and JT analyzed the data. XYW and GS discussed the results. XYW wrote the initial draft. XYW, XW, GS, YD, CL, and CKL substantially revised the manuscript. All authors agreed to be accountable for all aspects of this work.This work was supported by the Scientific Research Foundation for high-level talents of Beijing University of Chinese Medicine and the Beijing Technology Development of Traditional Chinese Medicine Foundation–Annual Planning Project (ref: JJ 2013-40).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.