miRNA-mediated regulation of clock gene expression in men and women with colorectal cancer and possible consequences for disease management.

BACKGROUND: The incidence and mortality of colorectal cancer (CRC) are persistently higher in men than in women. CRC malignancy is strongly influenced by small non-coding RNAs (miRNAs). Moreover, deregulation of the circadian molecular oscillator has been associated with CRC facilitation. To analyse possible cumulative effects of the above-mentioned factors on CRC progression, we focused on functions of sex-biased miRNAs associated with the clock genes per2 and/or cry2, which are involved in the cell cycle control and DNA damage response. MAJOR FINDINGS: We identified miR-24, miR-92a, miR-181a, and miR-21 associated with per2 that are up-regulated in transformed colon tissue of men. miR-93, miR-17, miR-20a, and miR-24 with higher expression in males compared to females were linked to cry2. All these miRNAs possess oncogenic potential and exert their effects mainly via inhibition of the tumour suppressors phosphatase and tensin homolog (PTEN) and/or p53. Down-regulation of PTEN and p53 in men was further strengthened by inhibition of tumour suppressor per2. Oncogenic up-regulated miRNAs associated with per2 or cry2 in the transformed colon tissue of women were not detected. CONCLUSION: We conclude that the cancer-promoting, sex-biased miRNAs miR-24, miR-92a, miR-181a, miR-93, miR-17, miR-20a, and miR-21 associated with clock genes per2 and/or cry2 can contribute to the sex-dependent development of CRC via inhibition of the PTEN and p53 pathways.

Impact of Long-Lasting Environmental Factors on Regulation Mediated by the miR-34 Family.

The present review focuses on the interactions of newly emerging environmental factors with miRNA-mediated regulation. In particular, we draw attention to the effects of phthalates, electromagnetic fields (EMFs) and a disrupted light/dark cycle. miRNAs are small non-coding RNA molecules with a tremendous regulatory impact, which is usually executed via gene expression inhibition. To address the capacity of environmental factors to influence miRNA-mediated regulation, the miR-34 family was selected for its well-described oncostatic and neuro-modulatory properties. The expression of miR-34 is in a tissue-dependent manner to some extent under the control of the circadian system. There is experimental evidence implicating that phthalates, EMFs and the circadian system interact with the miR-34 family, in both lines of its physiological functioning. The inhibition of miR-34 expression in response to phthalates, EMFs and light contamination has been described in cancer tissue and cell lines and was associated with a decline in oncostatic miR-34a signalling (decrease in p21 expression) and a promotion of tumorigenesis (increases in Noth1, cyclin D1 and cry1 expressions). The effects of miR-34 on neural functions have also been influenced by phthalates, EMFs and a disrupted light/dark cycle. Environmental factors shifted the effects of miR-34 from beneficial to the promotion of neurodegeneration and decreased cognition. Moreover, the apoptogenic capacity of miR-34 induced via phthalate administration in the testes has been shown to negatively influence germ cell proliferation. To conclude, as the oncostatic and positive neuromodulatory functions of the miR-34 family can be strongly influenced by environmental factors, their interactions should be taken into consideration in translational medicine.

Effect of miR-34a on the expression of clock and clock-controlled genes in DLD1 and Lovo human cancer cells with different backgrounds with respect to p53 functionality and 17ß-estradiol-mediated regulation.

The small non-coding RNA miR-34a is a p53-regulated miRNA that acts as a tumour suppressor of colorectal cancer (CRC). Oncogenesis is also negatively influenced by deregulation of the circadian system in many types of tumours with various genetic backgrounds. As the clock gene per2 was recently recognized as one of the target genes of miR-34a, we focused on the miR-34a-mediated influence on the circadian oscillator in CRC cell lines DLD1 and LoVo, which differ in their p53 status. Previously, a sex-dependent association between the expression of per2 and that of miR-34a was demonstrated in CRC patients. Therefore, we also investigated the effect of 17ß-estradiol (E2) on miR-34a oncostatic functions. miR-34a mimic caused a pronounced inhibition of per2 expression in both cell lines. Moreover, miR-34a mimic significantly inhibited bmal1 expression in LoVo and rev-erbα expression in DLD1 cells and induced clock gene expression in both cell lines. miR-34a mimic caused a pronounced decrease in sirt1 and cyclin D1 expression, which may be related to the inhibition of proliferation observed after mir-34a administration in DLD1 cells. E2 administration inhibited the migration and proliferation of DLD1 cells. E2 and miR-34a, when administered simultaneously, did not potentiate each other's effects. To conclude, miR-34a strongly influences the expression of components of the circadian oscillator without respect to p53 status and exerts its oncostatic effects via inhibition of sirt1 and cyclin D1 mRNA expression. E2 administration inhibits the growth of DLD1 cells; however, this effect seems to be independent of miR-34a-mediated action. With respect to the possible use of miR-34a in cancer treatment, clock genes can be considered as off-target genes, as changes in their expression induced by miR-34a treatment do not contribute to the oncostatic functions of miR-34a. Possible ambiguous oncogenic characteristics should be taken into consideration in future clinical studies focused on miR-34a.

2.4 GHz Electromagnetic Field Influences the Response of the Circadian Oscillator in the Colorectal Cancer Cell Line DLD1 to miR-34a-Mediated Regulation.

Radiofrequency electromagnetic fields (RF-EMF) exert pleiotropic effects on biological processes including circadian rhythms. miR-34a is a small non-coding RNA whose expression is modulated by RF-EMF and has the capacity to regulate clock gene expression. However, interference between RF-EMF and miR-34a-mediated regulation of the circadian oscillator has not yet been elucidated. Therefore, the present study was designed to reveal if 24 h exposure to 2.4 GHz RF-EMF influences miR-34a-induced changes in clock gene expression, migration and proliferation in colorectal cancer cell line DLD1. The effect of up- or downregulation of miR-34a on DLD1 cells was evaluated using real-time PCR, the scratch assay test and the MTS test. Administration of miR-34a decreased the expression of per2, bmal1, sirtuin1 and survivin and inhibited proliferation and migration of DLD1 cells. When miR-34a-transfected DLD1 cells were exposed to 2.4 GHz RF-EMF, an increase in cry1 mRNA expression was observed. The inhibitory effect of miR-34a on per2 and survivin was weakened and abolished, respectively. The effect of miR-34a on proliferation and migration was eliminated by RF-EMF exposure. In conclusion, RF-EMF strongly influenced regulation mediated by the tumour suppressor miR-34a on the peripheral circadian oscillator in DLD1 cells.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

miRNA Clusters with Up-Regulated Expression in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignancies in Europe and North America. Early diagnosis is a key feature of efficient CRC treatment. As miRNAs can be used as CRC biomarkers, the aim of the present study was to analyse experimentally validated data on frequently up-regulated miRNA clusters in CRC tissue and investigate their members with respect to clinicopathological characteristics of patients. Based on available data, 15 up-regulated clusters, miR-106a/363, miR-106b/93/25, miR-17/92a-1, miR-181a-1/181b-1, miR-181a-2/181b-2, miR-181c/181d, miR-183/96/182, miR-191/425, miR-200c/141, miR-203a/203b, miR-222/221, mir-23a/27a/24-2, mir-29b-1/29a, mir-301b/130b and mir-452/224, were selected. The positions of such clusters in the genome can be intronic or intergenic. Most clusters are regulated by several transcription factors, and miRNAs are also sponged by specific long non-coding RNAs. In some cases, co-expression of miRNA with other cluster members or host gene has been proven. miRNA expression patterns in cancer tissue, blood and faeces were compared. Based on experimental evidence, 181 target genes of selected clusters were identified. Panther analysis was used to reveal the functions of the target genes and their corresponding pathways. Clusters miR-17/92a-1, miR-106a/363, miR-106b/93/25 and miR-183/96/182 showed the strongest association with metastasis occurrence and poor patient survival, implicating them as the most promising targets of translational research.

miRNA Clusters with Down-Regulated Expression in Human Colorectal Cancer and Their Regulation.

Regulation of microRNA (miRNA) expression has been extensively studied with respect to colorectal cancer (CRC), since CRC is one of the leading causes of cancer mortality worldwide. Transcriptional control of miRNAs creating clusters can be, to some extent, estimated from cluster position on a chromosome. Levels of miRNAs are also controlled by miRNAs "sponging" by long non-coding RNAs (ncRNAs). Both types of miRNA regulation strongly influence their function. We focused on clusters of miRNAs found to be down-regulated in CRC, containing miR-1, let-7, miR-15, miR-16, miR-99, miR-100, miR-125, miR-133, miR-143, miR-145, miR-192, miR-194, miR-195, miR-206, miR-215, miR-302, miR-367 and miR-497 and analysed their genome position, regulation and functions. Only evidence provided with the use of CRC in vivo and/or in vitro models was taken into consideration. Comprehensive research revealed that down-regulated miRNA clusters in CRC are mostly located in a gene intron and, in a majority of cases, miRNA clusters possess cluster-specific transcriptional regulation. For all selected clusters, regulation mediated by long ncRNA was experimentally demonstrated in CRC, at least in one cluster member. Oncostatic functions were predominantly linked with the reviewed miRNAs, and their high expression was usually associated with better survival. These findings implicate the potential of down-regulated clusters in CRC to become promising multi-targets for therapeutic manipulation.

Expression of miR-34a-5p is up-regulated in human colorectal cancer and correlates with survival and clock gene PER2 expression.

Colorectal cancer represents a leading cause of cancer death. MicroRNAs (miRNAs) are small non-coding RNA molecules that have been extensively studied in tumours, since changes in their levels can reveal patient prognosis. Cancer progression is also influenced by the circadian system whose functioning is based on the rhythmic expression of clock genes. Therefore, we performed macroarray screening of tumour and adjacent tissues in patients undergoing surgery for colorectal carcinoma. We identified 17 miRNAs showing expression that was more than 100 times higher in tumour tissue compared to adjacent tissue. From in silico analysis, miR-34a-5p was selected as showing a computer-predicted interaction with PER2. Real-time PCR revealed a negative correlation between expression of PER2 mRNA and miR-34a in patients with more advanced cancer stage. Expression of miR-34a was up-regulated in cancer tissue compared to adjacent tissue. High miR-34a expression was associated with better survival of patients. miR-34a showed lower expression levels in male patients with lymph node involvement, and a trend towards decreased expression in male patients with distant metastases. Male patients, but not female patients, with high expression of miR-34a and who were free of distant metastases and/or lymph node involvement showed better survival. Therefore, we proposed that expression of miR-34a was regulated in a sex-dependent manner and could be considered a marker of prognosis in earlier cancer stages in male patients.

Sex and salt intake dependent renin-angiotensin plasticity in the liver of the rat.

OBJECTIVE: Epidemiological studies confirm that hypertensive patients respond differently to renin-angiotensin system (RAS) inhibition depending on their gender. The aim of present work is to focus on sex-dependent differences in RAS regulation under conditions of increased salt intake. METHOD: To investigate RAS, we measured the expression of angiotensinogen (Agt) mRNA, angiotensin receptor type 1 (AT1) mRNA and mitochondria assembly receptor (MasR) in the liver of rats under control conditions and after feeding with a salt diet (2% NaCl). In parallel, vascular endothelial growth factor A (VEGF-A) mRNA was analyzed. RESULTS: Regression analysis revealed sex-dependent differences in the correlation between mRNA expression of AT1 and that of Agt, MasR and VEGF-A in both groups. There was a significant negative correlation between AT1 and Agt mRNA expression in the male control group, but this correlation disappeared in males exposed to a salt diet. In females, AT1 and Agt expression correlated only in the group exposed to the salt diet. In control males, there was a borderline trend to correlation between AT1 and MasR mRNA expression. The correlation between AT1 and VEGF-A mRNA expression was significant only in the control females, however, after exposure to a salt diet, this correlation diminished. CONCLUSIONS: We hypothesize that RAS components expression is compensated differently in males and females. The observed loss of compensatory relationships in RAS between AT1 and Agt and AT1 and MasR in male rats under a salt diet can contribute to the differences observed in human with hypertension associated with an unhealthy diet.

Sex-dependent correlation between survival and expression of genes related to the circadian oscillator in patients with colorectal cancer.

Recent evidence supports the important role of the circadian system in cancer progression in humans. The aim of the present study is to evaluate clock (cry1, cry2 and per2) and clock-controlled (vascular endothelial growth factor-a, early growth response protein 1 and estrogen receptor ß) gene expression in colorectal cancer and adjacent tissue and identify a possible link between survival of patients and expression of above mentioned genes. The study includes 64 patients of both sexes with previously diagnosed colorectal cancer. RNA was extracted from the tumor tissue and adjacent parts of the resected colon, and real-time PCR was used for detection of clock gene expression. Expression of cry2 and per2 was significantly downregulated in tumor tissue compared to adjacent tissues. After splitting of the cohort according to sex, we detected downregulated levels of cry2 and per2 in male patients, but not in females. Splitting of male and female sub-cohorts according to presence of metastases revealed significant donwregulation of cry2 expression in female patients without distant metastasis. Better survival rate was associated with low expression of cry2 in female patients. Moreover, we observed an increase in cry1 expression in female patients with distant metastases in tumor compared to adjacent tissue. Accordingly, women with high expression of cry1 in tumor tissue displayed worse survival, which was not observed in men. Taken together, expression of clock and clock-controlled genes in tumors of males and females clustered according to presence of distant metastases correlated with survival analysis. Studied clock-controlled genes also showed sex-dependent changes. Low expression of vegf-a in tumor correlated with better survival in men but not in women. High expression of estrogen receptor ß mRNA was related to better survival in women but not in men. Low expression of vegf-a, egr1 and estrogen receptor ß was associated with worse survival in women compared to men. Our data indicate sex-dependent associations between clock and clock-controlled gene expression in cancer tissue and patient's survival prognosis.

Angiotensin II in the Human Physiology: Novel Ways for Synthetic Compounds Utilization.

BACKGROUND: Renin-angiotensin system (RAS) and its main product Angiotensin II (AngII) are in the focus of the pharmacological industry mainly because of hypertension treatment. Up-regulated RAS is generally associated with cardiovascular diseases and consequent organs injuries. The classic inhibition of RAS is based on the blocking of the type 1 AngII receptors and inhibition of ACE. The concept of the circulating and tissue RAS opens new challenges for the drug targeting. In spite of a big effort invested, in some cases a traditional RAS manipulation is struggling with unwanted side effects and/or resistance to treatment. OBJECTIVE: To improve the efficiency of the classic RAS inhibitors specific complications issuing from feed-back circuits inside the RAS have to be elucidated. Moreover, new peptidases identified in the AngII biosynthesis and Angiotensin 1-7/MAS pathways with opposing effects to AngII are tested for the clinical use. The aim of this review is also to bring attention to new tools in RAS manipulation based on the RNA interference (RNAi). RNAi employs small non-coding nucleic acids that interfere with the mRNA translation. The usefulness of this approach has been demonstrated in the treatment of oncological diseases and progress was also made in the field of the cardiovascular medicine. CONCLUSION: We suppose that in the near future, in addition to traditional pharmacological tools, RNAi will contribute to the control of RAS and AngII production. RNAi may also be of importance in the manipulation of tissue RAS that is not easily accessible by the traditional chemical substances.

Micro RNAs: an arguable appraisal in medicine.

Micro RNAs (miRNAs) represent a newly discovered class of regulatory molecules in the human body. miRNA is a short double stranded RNA sequence interfering with mRNA, causing in most cases, inhibition of translation. Synthesis of miRNAs shows an increasing developmental pattern and postnatally miRNAs are synthesized in all cells possessing transcriptional machinery. miRNAs usually target several mRNAs and therefore conclusive evidences proving their functions are not always ease to be acquired. In spite of this difficulty, functions of miRNAs were firmly established in the development, the cardiovascular and neural diseases, and cancer. Many miRNAs have been reported to be associated with physiological state of cells and/or tissues. This finding becomes fundamental, especially when consider that these miRNAs can be released from cell into intracellular space or circulation. Correlation between miRNA production in tissues and its contribution to multisource miRNA pool in the circulation is in a focus of biomarker-oriented researchers. Recently, circulating miRNAs have been suggested to be applicable as biomarkers in several types of cancer, cardiovascular injury, and diabetes. Role of miRNAs in the organism intercellular signaling is still under the broad investigation. Several miRNA mimics, intended for treatment of disease, are being currently tested in the clinical trials.

Expression of cell cycle regulatory factors hus1, gadd45a, rb1, cdkn2a and mre11a correlates with expression of clock gene per2 in human colorectal carcinoma tissue.

Deregulated expression of clock gene per2 has previously been associated with progression of cancer. The aim of the present study was to identify genes related to per2 expression and involved in cell cycle control. Patients surgically treated for colorectal carcinoma with up-regulated and down-regulated per2 expression in cancer versus adjacent tissue were studied. Total RNA from cancer tissue of these patients was used to specify genes associated with altered per2 expression using the Human Cell Cycle RT(2) profiler PCR array system. We identified seven genes positively correlated (hus1, gadd45α, rb1, cdkn2a, cdk5rp1, mre11a, sumo1) and two genes negatively correlated (cdc20, birc5) with per2 expression. Expression of these seven genes was subsequently measured by real time PCR in all patients of the cohort. Patients were divided into three groups according to TNM classification. We observed an increase in gene expression in cancer tissue compared to adjacent tissue in the first group of patients in all genes measured. Expression of genes positively associated with per2 gene expression was dependent on tumor staging and changes were observed preferentially in cancer tissue. For genes negatively associated with per2 expression we also detected changes in expression dependent on tumor staging. Expression of cdc20 and birc5 was increasing in the proximal tissue and decreasing in the cancer tissue. These results implicate functional involvement of per2 in the process of carcinogenesis via newly uncovered genes. The relevancy of gene expression for determination of diagnosis and prognosis should be considered in relation to tumor staging.

Deregulated expression of the per2 gene in human colorectal carcinoma.

The circadian system is involved in the control of cell proliferation and apoptosis. The aim of this study was to analyze expression of the human per2 gene in patients who underwent surgery for colorectal carcinoma. The study included 25 patients of both genders. Patients were exposed to light from 6:00 a.m. until 9:00 p.m. according to standard hospital practice. Tissue samples were taken from the tumor as well as from the proximal and distal areas of the resected colon at the time of surgery. Surgery was performed during the morning hours. Expression of per2 mRNA was measured by real-time PCR. There was a significant negative correlation between per2 gene expression and tumor staging. Expression of per2 mRNA did not correlate with whether the tumor was localized in the colon or rectum. In comparison with ectomized tissue without malignancy from patients with colorectal carcinoma, our data demonstrate per2 mRNA deregulation in tumor tissue, and suggest a way in which the circadian system can influence tumorigenesis.

Evidence for daily rhythms of the expression of proopiomelanocortin, interleukin-1-beta and interleukin-6 in adenopituitaries of male long-evans rats: effect of adjuvant arthritis.

OBJECTIVE: Several interleukins (ILs) including IL-1 beta and IL-6 are produced in the anterior pituitary (AP) where they probably participate in the local regulation of hormone production. Immune challenge brings about the dysregulation of immune-endocrine interaction and enhanced the expression of pituitary IL-1 beta and IL-6. Little is known about regulation of their production, and therefore the purpose of the present work was to describe the relationship between circulating corticosterone and the mRNA expression of proopiomelanocortin (POMC), IL-1 beta and IL-6 in the AP during a 24-hour cycle in normal rats and rats with acute adjuvant arthritis (AA). METHODS: Groups of intact male Long-Evans rats and rats 23 days after induction of AA kept on a 12-hour light/dark cycle (light on at 6:00 a.m.) were killed at 4-hour intervals starting at 2:00 p.m. Trunk blood was used for corticosterone determination by radioimmunoassay. Adenopituitaries were extracted for total RNA and the message of interest was quantitated by real-time PCR using specific primers and TaqMan probes. Parameters of rhythms were evaluated by cosinor analysis. RESULTS: In normal rats, serum corticosterone showed a circadian rhythm with the peak at 6:00 p.m. and the nadir in the morning hours (p < 0.001). POMC mRNA in AP also showed a circadian rhythm (p < 0.05) which was inversely related to corticosterone levels. IL-1beta and IL-6 expression in normal rats showed clear-cut daily rhythms (p < 0.001) with the nadirs in the dark period, in contrast to the corticosterone peak in plasma. In arthritic rats, rhythmic corticosterone secretion was suppressed with a plateau pattern of the rhythm. The mean POMC expression was higher than in controls, and the rhythm failed to be significant. IL-1 beta expression was suppressed by AA (p < 0.001) but the rhythm was still present (p < 0.05). The rhythmic pattern of IL-6 expression was similar to that of controls, but with higher mesor values (p < 0.05). CONCLUSION: These results suggest a regulatory relationship between circulating corticosterone and the expression of POMC, IL-1 beta and IL-6 in AP of normal rats. Arthritis induced a higher expression of POMC and IL-6 in the AP and a suppression of IL-1 beta mRNA during the 24-hour cycle which suggests the involvement of different regulatory mechanisms compared to normal conditions.

Daily profiles of plasma prolactin (PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), testosterone, and melatonin, and of pituitary PRL mRNA and GH mRNA in male Long Evans rats in acute phase of adjuvant arthritis.

We studied the effects of adjuvant arthritis (AA) on the endocrine circadian rhythms of plasma prolactin (PRL), growth hormone (GH), insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), testosterone, and melatonin and of pituitary PRL and GH mRNA in male Long Evans rats. Groups of control and AA rats (studied 23 days after AA induction) that were housed under a 12/12 h light/dark cycle (light on at 06:00 h) were killed at 4 h intervals starting at 14:00 h. Cosinor analysis revealed a significant 12 h rhythm in PRL and PRL mRNA (p < 0.001) in controls with peaks at 14:00 h and 02:00 h, respectively. The peak at 02:00 h was abolished in the AA group resulting in a significant 24 h rhythm in parallel with that of PRL (p < 0.05) and PRL mRNA (p < 0.0001). Growth hormone showed no rhythm, but a significant rhythm of GH mRNA was present in both groups (p < 0.0001). Insulin-like growth factor-1 showed a 24 h rhythm in control but not in AA rats. The mean values of GH, GH mRNA, and IGF-1 were significantly reduced in AA. Luteinizing hormone displayed a significant 24 h rhythm (p < 0.01) peaking in the dark period in the control but not AA group. Testosterone showed in phase temporal changes of LH levels with AA abolishing the 02:00 h peak. Melatonin exhibited a significant 24 h rhythm in control (p < 0.001) and AA (p < 0.01) rats with maximum levels during the dark phase; the mesor value was higher in the AA males. These results demonstrate that AA interferes with the rhythms of all the studied hormones except the non-24 h (arrhythmic) GH secretion pattern and the rhythm in melatonin. The persistence of a distinct melatonin rhythm in AA suggests the observed disturbances of hormonal rhythms in this condition do not occur at the level of the pineal gland.