Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects.

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.

Assessment of Epiregulin Effect and its Combination with Gonadotropins on Proliferation, Apoptosis, and Secretory Activity by Human Ovarian Cells.

The release of epidermal growth factor ligand epiregulin (EREG) by human ovarian granulosa cells, its direct action on basic ovarian cell functions, and interrelationships with gonadotropins were investigated. We examined (1) the ovarian production of EREG (the time-dependent accumulation of EREG in the medium incubated with human ovarian granulosa cells, and (2) the effect of the addition of EREG (0, 1, 10, and 100 ng.ml-1) given alone or in combination with FSH or LH (100 ng.ml-1) on basic granulosa cells functions. Viability, proliferation (accumulation of PCNA and cyclin B1) and apoptosis (accumulation of bax and caspase 3), the release of steroid hormones (progesterone, testosterone, and estradiol), and prostaglandin E2 (PGE2) were analyzed by using the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. A significant time-dependent accumulation of EREG in a medium cultured with human granulosa cells with a peak at 3 and 4 days was observed. The addition of EREG alone increased cell viability, proliferation, progesterone, testosterone, and estradiol release, decreased apoptosis, bud did not affect PGE2 release. The addition of either FSH or LH alone increased cell viability, proliferation, progesterone, testosterone, estradiol, and PGE2 release and decreased apoptosis. Furthermore, both FSH and LH mostly promoted the stimulatory action of EREG on granulosa cell functions. These results demonstrated, that EREG produced by ovarian cells can be an autocrine/paracrine stimulator of human ovarian cell functions. Furthermore, they demonstrate the functional interrelationship between EREG and gonadotropins in the control of ovarian functions.

Direct action of leptin, obestatin and ginkgo on hormone release by luteinised human ovarian granulosa cells.

CONTEXT: The role of metabolic hormones, medicinal plants and their interrelationships in the control of human reproductive processes are poorly understood. AIMS: To examine how leptin, obestatin and ginkgo (Ginkgo biloba L.) affect human ovarian hormone release. METHODS: We analysed the influence of leptin and obestatin alone and in combination with ginkgo extract on cultured human ovarian granulosa cells. The release of progesterone (P), insulin-like growth factor I (IGF-I), oxytocin (OT) and prostaglandin F (PGF) were analysed by enzyme immunoassay and enzyme-linked immunosorbent assay. KEY RESULTS: Leptin addition promoted the release of all the measured hormones. Obestatin stimulated the release of P, IGF-I and OT and inhibited PGF output. Ginkgo suppressed P, IGF-I and OT and promoted PGF release. Furthermore, ginkgo changed the stimulatory action of leptin on PGF to an inhibitory one. CONCLUSIONS: Leptin and obestatin are involved in the control of human ovarian hormone release and ginkgo influences their function. IMPLICATIONS: Leptin and obestatin could be useful as stimulators of human ovarian cell functions. The suppressive influence of ginkgo on ovarian function should lead to the development of ginkgo-containing drugs.

The Joint Effect of Perceived Psychosocial Stress and Phthalate Exposure on Hormonal Concentrations during the Early Stage of Pregnancy: A Cross-Sectional Study.

Phthalates alter the hormonal balance in humans during pregnancy, potentially affecting embryonic and fetal development. We studied the joint effect of exposure to phthalates, quantified by urinary phthalate metabolite concentration, and perceived psychological stress on the concentration of hormones in pregnant women (n = 90) from the Nitra region, Slovakia, up to the 15th week of pregnancy. We used high-performance liquid chromatography, tandem mass spectrometry (HPLC-MS/MS), and electro-chemiluminescence immunoassay to determine urinary concentrations of phthalates and serum concentrations of hormones, respectively. We used Cohen perceived stress scale (PSS) to evaluate the human perception of stressful situations. Our results showed that mono(carboxy-methyl-heptyl) phthalate (cx-MiNP) and a molar sum of di-iso-nonyl phthalate metabolites (ΣDiNP) were negatively associated with luteinizing hormone (LH) (p ≤ 0.05). Mono(hydroxy-methyl-octyl) phthalate (OH-MiNP) and the molar sum of high-molecular-weight phthalate metabolites (ΣHMWP) were positively associated with estradiol (p ≤ 0.05). PSS score was not significantly associated with hormonal concentrations. When the interaction effects of PSS score and monoethyl phthalate (MEP), cx-MiNP, ΣDiNP, and ΣHMWP on LH were analyzed, the associations were positive (p ≤ 0.05). Our cross-sectional study highlights that joint psychosocial stress and xenobiotic-induced stress caused by phthalates are associated with modulated concentrations of reproductive hormones in pregnant women.

Kisspeptin as autocrine/paracrine regulator of human ovarian cell functions: Possible interrelationships with FSH and its receptor.

The present study aims to examine the role of kisspeptin (KP), FSH, and its receptor (FSHR), and their interrelationships in the control of basic human ovarian granulosa cells functions. We investigated: (1) the ability of granulosa cells to produce KP and FSHR, (2) the role of KP in the control of ovarian functions, and (3) the ability of KP to affect FSHR and to modify the FSH action on ovarian functions. The effects of KP alone (0, 10 and 100 ng/mL); or of KP (10 and 100 ng/mL) in combination with FSH (10 ng/mL) on cultured human granulosa cells were assessed. Viability, markers of proliferation (PCNA and cyclin B1) and apoptosis (bax and caspase 3), as well as accumulation of KP, FSHR, and steroid hormones, IGF-I, oxytocin (OT), and prostaglandin E2 (PGE2) release were analyzed by the Trypan blue exclusion test, quantitative immunocytochemistry, and ELISA. KP given at a low dose (10 ng/mL) stimulated viability, proliferation, inhibited apoptosis, promoted the release of progesterone (P4), estradiol (E2), IGF-I, OT, and PGE2, the accumulation of FSHR, but not testosterone (T) release. KP given at a high dose (100 ng/mL) had the opposite, inhibitory effect. FSH stimulated cell viability, proliferation and inhibited apoptosis, promoted P4, T, E2, IGF-I, and OT, but not PGE2 release. Furthermore, KP at a low dose promoted the stimulatory effect of FSH on viability, proliferation, P4, E2, and OT release, promoted its inhibitory action on apoptosis, but did not modify its action on T, IGF-I, and PGE2 output. KP at a high dose prevented and inverted FSH action. These results suggest an intra-ovarian production and a functional interrelationship between KP and FSH/FSHR in direct regulation of basic ovarian cell functions (viability, proliferation, apoptosis, and hormones release). The capability of KP to stimulate FSHR, the ability of FSH to promote ovarian functions, as well as the similarity of KP (10 ng/mL) and FSH action on granulosa cells' viability, proliferation, apoptosis, steroid hormones, IGF-I, OT, and PGE2 release, suggest that FSH influence these cells could be mediated by KP. Moreover, the capability of KP (100 ng/mL) to decrease FSHR accumulation, basal and FSH-induced ovarian parameters, suggest that KP can suppress some ovarian granulosa cell functions via down-regulation of FSHR. These observations propose the existence of the FSH-KP axis up-regulating human ovarian cell functions.

Cold Atmospheric Pressure Plasma (CAP) as a New Tool for the Management of Vulva Cancer and Vulvar Premalignant Lesions in Gynaecological Oncology.

Vulvar cancer (VC) is a specific form of malignancy accounting for 5-6% of all gynaecologic malignancies. Although VC occurs most commonly in women after 60 years of age, disease incidence has risen progressively in premenopausal women in recent decades. VC demonstrates particular features requiring well-adapted therapeutic approaches to avoid potential treatment-related complications. Significant improvements in disease-free survival and overall survival rates for patients diagnosed with post-stage I disease have been achieved by implementing a combination therapy consisting of radical surgical resection, systemic chemotherapy and/or radiotherapy. Achieving local control remains challenging. However, mostly due to specific anatomical conditions, the need for comprehensive surgical reconstruction and frequent post-operative healing complications. Novel therapeutic tools better adapted to VC particularities are essential for improving individual outcomes. To this end, cold atmospheric plasma (CAP) treatment is a promising option for VC, and is particularly appropriate for the local treatment of dysplastic lesions, early intraepithelial cancer, and invasive tumours. In addition, CAP also helps reduce inflammatory complications and improve wound healing. The application of CAP may realise either directly or indirectly utilising nanoparticle technologies. CAP has demonstrated remarkable treatment benefits for several malignant conditions, and has created new medical fields, such as "plasma medicine" and "plasma oncology". This article highlights the benefits of CAP for the treatment of VC, VC pre-stages, and postsurgical wound complications. There has not yet been a published report of CAP on vulvar cancer cells, and so this review summarises the progress made in gynaecological oncology and in other cancers, and promotes an important, understudied area for future research. The paradigm shift from reactive to predictive, preventive and personalised medical approaches in overall VC management is also considered.

Rho GTPases in Gynecologic Cancers: In-Depth Analysis toward the Paradigm Change from Reactive to Predictive, Preventive, and Personalized Medical Approach Benefiting the Patient and Healthcare.

Rho guanosine triphospatases (GTPases) resemble a conserved family of GTP-binding proteins regulating actin cytoskeleton dynamics and several signaling pathways central for the cell. Rho GTPases create a so-called Ras-superfamily of GTPases subdivided into subgroups comprising at least 20 members. Rho GTPases play a key regulatory role in gene expression, cell cycle control and proliferation, epithelial cell polarity, cell migration, survival, and apoptosis, among others. They also have tissue-related functions including angiogenesis being involved in inflammatory and wound healing processes. Contextually, any abnormality in the Rho GTPase function may result in severe consequences at molecular, cellular, and tissue levels. Rho GTPases also play a key role in tumorigenesis and metastatic disease. Corresponding mechanisms include a number of targets such as kinases and scaffold/adaptor-like proteins initiating GTPases-related signaling cascades. The accumulated evidence demonstrates the oncogenic relevance of Rho GTPases for several solid malignancies including breast, liver, bladder, melanoma, testicular, lung, central nervous system (CNS), head and neck, cervical, and ovarian cancers. Furthermore, Rho GTPases play a crucial role in the development of radio- and chemoresistance e.g. under cisplatin-based cancer treatment. This article provides an in-depth overview on the role of Rho GTPases in gynecological cancers, highlights relevant signaling pathways and pathomechanisms, and sheds light on their involvement in tumor progression, metastatic spread, and radio/chemo resistance. In addition, insights into a spectrum of novel biomarkers and innovative approaches based on the paradigm shift from reactive to predictive, preventive, and personalized medicine are provided.

Transcription factor p53 regulates healthy human ovarian cells function.

The aim of our study was to understand the role of transcription factor p53 in the control of healthy human ovarian cell functions. Ovarian granulosa cells were transfected with a cDNA construct encoding p53. The intracellular accumulation of p53, of the apoptosis marker bax, and of the proliferation marker PCNA, as well as the release of progesterone (P4), insulin-like growth factor I (IGF-I), oxytocin (OT), and prostaglandin F (PGF) and E2 (PGE) were evaluated by quantitative immunocytochemistry and RIA/IRMA. Transfection with the p53 cDNA construct resulted in the accumulation of p53 and bax, in a reduced level of released PCNA and PGF, and in an increased PGE output. No changes in P4, IGF-I, and OT secretion were found. These observations are the first demonstration of the involvement of p53 in the control of healthy human ovarian cell functions, namely, in the downregulation of proliferation, in the upregulation of apoptosis, and in the alteration of PGF and PGE release, but not of P4, IGF-I, or OT.

Oncological outcome of surgical management in patients with recurrent uterine cancer-a multicenter retrospective cohort study-CEEGOG EX01 Trial.

OBJECTIVES: To assess the survival of patients who have received an operation for recurrent cervical and endometrial cancer and to determine prognostic variables for improved oncologic outcome. METHODS: A retrospective multicenter analysis of the medical records of 518 patients with cervical (N = 288) or endometrial cancer (N = 230) who underwent surgery for disease recurrence and who had completed at least 1 year of follow-up. RESULTS: The median survival reached 57 months for patients with cervical cancer and 113 months for patients with endometrial cancer after surgical treatment of recurrence (p = 0.036). Histological sub-type had a significant impact on overall survival, with the best outcome in endometrial endometrioid cancer (121 months), followed by cervical squamous cell carcinoma, cervical adenocarcinoma, or other types of endometrial cancer (81 vs 35 vs 35 months; p <0.001). The site of recurrence did not significantly influence survival in cervical or in endometrial cancer. Cancer stage at first diagnosis, tumor grade, lymph node status at recurrence, progression-free interval after first diagnosis, and free resection margins were associated with improved overall survival on univariate analysis. On multivariate analysis, the stage at first diagnosis and resection margins were significant independent predictive parameters of an improved oncologic outcome. CONCLUSION: Long-term survival can be achieved via secondary cytoreductive surgery in selected patients with recurrent cervical and endometrial cancer. An excellent outcome is possible even if the recurrence site is located in the lymph nodes. The possibility of achieving complete resection should be the main criterion for patient selection.

The involvement of the phosphorylatable and nonphosphorylatable transcription factor CREB-1 in the control of human ovarian cell functions.

The objective of our study was to elucidate the role of the transcription factor CREB-1 in controlling ovarian cell proliferation, apoptosis, and hormone release and the significance of CREB-1 phosphorylation in these processes. Human ovarian granulosa cells were transfected with a gene construct encoding wild-type CREB-1 (CREB-1 WT) or CREB-1 nonphosphorylatable mutant (CREB-1 M1). The expression of total and phosphorylated CREB-1, markers of proliferation (PCNA) and apoptosis (bax), as well as the release of progesterone, oxytocin, prostaglandin F2 alpha (PGF2), prostaglandin E2 (PGE2), and insulin-like growth factor I (IGF-I) were compared by immunocytochemistry, enzyme immunoassay (EIA), and immunoradiometric assay (IRMA). Transfection with CREB-1 WT or CREB-1 M1 increased total CREB-1 expression and proliferation and decreased the release of oxytocin, PGE2, and IGF-I by ovarian cells. CREB-1 M1, not CREB-1 WT, promoted apoptosis and inhibited progesterone output. PGF2 release was inhibited by CREB-1 WT but stimulated by CREB-1 M1 construct. Phosphorylated CREB-1 was undetected in any cell group. These observations confirm the involvement of CREB-1 in the control of ovarian cell proliferation, apoptosis, and steroid hormone release. This is the first demonstration of the involvement of CREB-1 in the regulation of the ovarian non-steroidal hormones such as oxytocin, PGF2, PGE2, and IGF-I. The absence of CREB-1 phosphorylation, similar effects exerted by CREB-1 WT and CREB-1 M1 on cell proliferation and release of oxytocin, PGE2, and IGF-I, and the influence of CREB-1 M1 on apoptosis and progesterone suggest that phosphorylation plays no role in the action of CREB-1 on the majority of analyzed functions of human ovarian cells.

Comparison of the effects of synthetic and plant-derived mTOR regulators on healthy human ovarian cells.

The aim of the present in vitro study was to compare the effects of synthetic and plant-derived mTOR regulators on healthy human ovarian cells. We compared the effect of two synthetic mammalian mTOR blockers MC2141 and MC2183 with that of natural/plant-derived mTOR blocker rapamycin and mTOR activator resveratrol on cultured human ovarian granulosa cells. We evaluated the accumulation of markers for the mTOR system (sirtuin 1; SIRT 1), proliferation (PCNA), and apoptosis (caspase 3) along with the expression of the transcription factor p53 by quantitative immunocytochemistry. It was observed that MC2183 but not MC2141 or rapamycin reduced SIRT 1 accumulation. MC2141, MC2183, and rapamycin inhibited the accumulation of PCNA, caspase 3, and p53. On the contrary, resveratrol promoted the accumulation of SIRT-1, PCNA, caspase 3, and p53. We have demonstrated the involvement of the mTOR system in the regulation of healthy human ovarian cell proliferation and apoptosis for the first time and indicated that the action of mTOR regulators on ovarian cell apoptosis can be mediated by p53. We have further shown that mTOR regulators can affect ovarian functions without any changes in SIRT-1 accumulation and that the stimulatory effects of resveratrol on analyzed ovarian cell functions are opposite to the inhibitory effects of rapamycin and synthetic mTOR blockers.

Dietary Phytochemicals Targeting Cancer Stem Cells.

There is an increasing awareness of the importance of a diet rich in fruits and vegetables for human health. Cancer stem cells (CSCs) are characterized as a subpopulation of cancer cells with aberrant regulation of self-renewal, proliferation or apoptosis leading to cancer progression, invasiveness, metastasis formation, and therapy resistance. Anticancer effects of phytochemicals are also directed to target CSCs. Here we provide a comprehensive review of dietary phytochemicals targeting CSCs. Moreover, we evaluate and summarize studies dealing with effects of dietary phytochemicals on CSCs of various malignancies in preclinical and clinical research. Dietary phytochemicals have a significant impact on CSCs which may be applied in cancer prevention and treatment. However, anticancer effects of plant derived compounds have not yet been fully investigated in clinical research.

MicroRNAs control transcription factor NF-kB (p65) expression in human ovarian cells.

MicroRNAs (miRNAs) are known to influence ovarian cell proliferation, apoptosis and hormone release, but it remains unknown whether miRNAs affect ovarian functions via transcription factors. We examined the effect of miRNAs on nuclear factor-κappaB (NF-kB) (p65) expression in human ovarian luteinized granulosa cells. We transfected cultured primary human ovarian luteinized granulosa cells with 80 different constructs encoding human pre-miRNAs and then evaluated NF-kB (p65) expression (percentage of cells containing p65) by immunocytochemistry. We found that 21 of the constructs stimulated NF-kB (p65) expression and 18 of the constructs inhibited NF-kB (p65) expression. This is the first direct demonstration that miRNAs affect NF-kB (p65) expression and the first genome-scale miRNA screen to identify upregulation and downregulation of NF-kB accumulation by miRNAs in the ovary. Novel miRNAs that affect the NF-kB signalling pathway could be useful for the control of NF-kB-dependent reproductive processes and the treatment of NF-kB-dependent reproductive disorders.

Involvement of microRNA Mir15a in control of human ovarian granulosa cell proliferation, apoptosis, steroidogenesis, and response to FSH.

Our study aimed to examine the role of micro RNA Mir15a in control of basic ovarian cell functions: proliferation, apoptosis, and secretory activity. In the first series of experiments, primary human ovarian granulosa cells were transfected with antisense construct blocking Mir15a (anti-Mir15a) and cultured without hormonal treatments. Accumulation of markers of proliferation (MAPK/ERK1,2 and PCNA) and apoptosis (caspase 3 and bax), and release of steroid hormones (progesterone, testosterone, and estradiol) were evaluated by immunocytochemical analysis and by enzyme immunoassay. In the second series of experiments, granulosa cells were transfected with gene construct encoding Mir15a precursor (pre-Mir15a) and cultured with and without follicle-stimulating hormone (FSH; 0, 1, 10, and 100 ng/ml). Expression of markers of proliferation (MAPK/ERK1,2) apoptosis (caspase 3), and steroidogenesis (release of progesterone, testosterone, and estradiol) were evaluated. Transfection of cells with anti-Mir15a resulted in a significant increase in accumulation of both proliferation and apoptosis markers, a reduction in progesterone and testosterone release, and an increase in estradiol release. Transfection of cells with pre-Mir15a had an opposite effect: it reduced accumulation of proliferation- and apoptosis-related proteins MAPK/ERK1,2 and caspase 3, and promoted release of progesterone and testosterone, but not estradiol. Moreover, pre-Mir15a reversed the effect of FSH on caspase 3, progesterone, and testosterone, but not on MAPK/ERK1,2 and estradiol. Our observations demonstrate involvement of Mir15a in control of multiple ovarian functions: proliferation, apoptosis, release of progesterone, androgen, and estrogen, and response to gonadotropin. Moreover, this is the first demonstration that miRNAs can affect response of cells to hormonal regulators. We propose that Mir15 could potentially be used for control of different reproductive processes.

Identification of microRNAs controlling human ovarian cell steroidogenesis via a genome-scale screen.

The aim of our studies was to identify miRNAs affecting the release of the major ovarian steroid hormones progestagen, androgen and estrogen by human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different gene constructs encoding human pre-miRNAs on release of progesterone, testosterone and estradiol was evaluated by enzyme immunoassay. In addition, effect of two selected antisense constructs blocking corresponding miRNA on progesterone release was tested. Efficiency of transfection (incorporation transfection reagent) and silencing of marker substances (GAPDH mRNA, GAPDH and CREB-1) were validated by fluorescent microscopy, real-time reverse transcription-PCR analysis and immunocytochemical analysis. Thirty-six out of 80 tested miRNA constructs resulted in inhibition of progesterone release in granulosa cells, and 10 miRNAs promoted progesterone release. Transfected of cells with antisense constructs to two selected miRNAs blocking progesterone release induced increase in progesterone output. Fifty-seven miRNAs tested inhibited testosterone release, and only one miRNA enhanced testosterone output. Fifty-one miRNAs suppressed estradiol release, while none of the miRNAs tested stimulated it. This is the first demonstration that miRNAs can control reproductive functions resulting in enhanced or inhibited release of ovarian progestagen, androgen and estrogen. We hypothesize that such miRNA-mediated effects could be potentially used for regulation of reproductive processes, including fertility, and for treatment of reproductive and other steroid-dependent disorders.

Protein kinases controlling PCNA and p53 expression in human ovarian cells.

The aim of this study was to identify protein kinases (PKs) involved in the expression of proliferating cell nuclear antigen (PCNA) and p53, markers of proliferation and apoptosis in human ovarian cells. Cultured ovarian granulosa cells were subjected to transfection with 264 small-interfering RNA (siRNA) constructs from a siRNA library, which selectively blocked the expression of 88 known PKs. The efficiency of transfection and siRNA knockdown were validated by fluorescent microscopy, real-time reverse transcription polymerase chain reaction, and immunocytochemical analysis. The expression of PCNA and p53, before and after transfection with siRNA constructs, was evaluated by immunocytochemistry. The siRNA constructs suppressed the expression of their targets molecules by up to 84%. Knockdown of 32 of the 88 PKs inhibited the expression of PCNA, while the knockdown of seven of the PKs stimulated PCNA expression. Knockdown of 30 of the 88 PKs reduced the expression of p53, while knockdown of five PKs enhanced p53 expression. Our results illustrate that siRNA constructs are useful tools for understanding the role of PKs in the control of ovarian cell functions, such as proliferation and apoptosis. The specific knockdown of individual PKs has enabled the identification of a number of new PKs that control the expression of PCNA and p53 in human ovarian cells.

Appendiceal adenocarcinoma presenting with bilateral Krukenberg tumors.

Primary appendiceal malignancy metastatic to the ovaries is a condition that may mimic advanced stage ovarian cancer. This condition is rarely diagnosed preoperatively. A 52-year-old woman referred to our institution for presumed advanced stage of ovarian cancer was found to have primary appendiceal adenocarcinoma metastatic to ovaries at laparotomy. We describe the clinical course of the patient. It is important for the gynecologist-oncologist to include tumors of the appendix into the differential diagnosis of any case of ovarian tumor.

Endometrial adenocarcinoma presenting with an osseous metastasis.

BACKGROUND: Gynecologic cancers metastatic to bone are rare. Endometrial carcinoma usually presents with vaginal bleeding. CASE REPORT: A 67-year-old woman presented with pain, erythema and swelling of the right foot and no history of postmenopausal bleeding. Biopsy revealed primary endometrioid carcinoma metastatic to the calcaneus, talus and metatarsal bones. Lower leg amputation, total abdominal hysterectomy, bilateral salpingo-oophorectomy and pelvic lymph node sampling were performed. Postoperatively the patient received cisplatin with adriamycin and megestrol acetate and is alive with no evidence of disease 20 months after the diagnosis. CONCLUSION: Endometrial carcinoma can present as a metastatic lesion of bone.