Automated Prognosis Marker Assessment in Breast Cancers Using BLEACH&STAIN Multiplexed Immunohistochemistry.

Prognostic markers in routine clinical management of breast cancer are often assessed using RNA-based multi-gene panels that depend on fluctuating tumor purity. Multiplex fluorescence immunohistochemistry (mfIHC) holds the potential for an improved risk assessment. To enable automated prognosis marker detection (i.e., progesterone receptor [PR], estrogen receptor [ER], androgen receptor [AR], GATA3, TROP2, HER2, PD-L1, Ki67, TOP2A), a framework for automated breast cancer identification was developed and validated involving thirteen different artificial intelligence analysis steps and an algorithm for cell distance analysis using 11+1-marker-BLEACH&STAIN-mfIHC staining in 1404 invasive breast cancers of no special type (NST). The framework for automated breast cancer detection discriminated normal glands from malignant glands with an accuracy of 98.4%. This approach identified that five (PR, ER, AR, GATA3, PD-L1) of nine biomarkers were associated with prolonged overall survival (p ≤ 0.0095 each) and two of these (PR, AR) were found to be independent risk factors in multivariate analysis (p ≤ 0.0151 each). The combined assessment of PR-ER-AR-GATA3-PD-L1 as a five-marker prognosis score showed strong prognostic relevance (p < 0.0001) and was an independent risk factor in multivariate analysis (p = 0.0034). Automated breast cancer detection in combination with an artificial intelligence-based analysis of mfIHC enables a rapid and reliable analysis of multiple prognostic parameters. The strict limitation of the analysis to malignant cells excludes the impact of fluctuating tumor purity on assay precision.

High Homogeneity of Mesothelin Expression in Primary and Metastatic Ovarian Cancer.

To study the extent of heterogeneity of mesothelin overexpression in primary ovarian cancers and their peritoneal and lymph node metastases, a tissue microarray (TMA) was constructed from multiple sites of 220 ovarian cancers and analyzed by immunohistochemistry. One tissue core each was taken from up to 18 different tumor blocks per cancer, resulting in a total of 2460 tissue spots from 423 tumor sites (188 primary cancers, 162 peritoneal carcinosis, and 73 lymph node metastases). Positive mesothelin expression was found in 2041 of the 2342 (87%) arrayed tissue spots and in 372 of the 392 (95%) tumor sites that were interpretable for mesothelin immunohistochemistry. Intratumoral heterogeneity was found in 23% of 168 primary cancer sites interpretable for mesothelin and decreased to 12% in 154 peritoneal carcinosis and to 6% in 71 lymph node metastases ( P <0.0001). Heterogeneity between the primary tumor and matched peritoneal carcinosis was found in 16% of 102 cancers with interpretable mesothelin results. In these cancers, the mesothelin status switched from positive in the primary tumor to negative in the peritoneal carcinosis (3 cancers) in or vice versa (2 cancers), or a mixture of positive and negative peritoneal carcinoses was found (11 cancers). No such switch was seen between the mesothelin-interpretable primary tumors and their nodal metastases of 59 cancers, and only 1 mesothelin-positive tumor had a mixture of positive and negative lymph node metastases. In conclusion, mesothelin expression is frequent and highly homogeneous in ovarian cancer.

Localization of claudin-2 and claudin-3 in eutopic and ectopic endometrium is highly similar.

PURPOSE: Claudins as the major components of tight junctions are important in maintaining cell-cell integrity and thus function as a barrier. Dysregulation of the claudins is often associated with loss of the epithelial phenotype, a process called epithelial-mesenchymal transition (EMT), which most often results in gain of migrative and invasive properties. However, the role of claudins in the endometrium or endometriosis has only rarely been examined. METHODS: In this study, we investigated localization of claudin-2 and claudin-3 in the eutopic and ectopic endometrium with immunohistochemistry. A detailed quantification with HSCORE was performed for claudin-2 and claudin-3 in endometrium without endometriosis and in cases with endometriosis compared to the three endometriotic entities: peritoneal, ovarian, and deep-infiltrating endometriosis. RESULTS: We found a preferential localization of both claudins in the glandular and the luminal epithelial cells in the endometrium with and without endometriosis. Quantification of localization of both claudins showed no differences in eutopic endometrium of control cases compared to cases with endometriosis. Furthermore, both claudins are localized highly similar in the ectopic compared to the eutopic endometrium, which is in clear contrast to previously published data for claudin-3. CONCLUSION: From our results, we conclude that localization of claudin-2 and claudin-3 is highly stable in eutopic and ectopic endometrium without any loss of the epithelial phenotype and thus do not contribute to the pathogenesis of endometriosis.

Acceptance of apitherapeutic methods in patients consulting general physicians or gynaecologists.

BACKGROUND: and purpose: Apitherapists promote the medical use of products from the beehive (bee venom, propolis, pollen, honey, royal jelly, dead bees, apilarnil, wax, wax moths), the use of beehive air or therapeutic sleep on a beehive. However, little is known about how far such treatment options are perceived as acceptable by patients. METHODS: Patients visiting either a family doctor in Kehl or a gynecologist in Weilburg (both in Germany) were asked to rate their knowledge of apitherapy as well as their readiness to use apitherapeutic measures. RESULTS: Honey and propolis represent the best-known bee products whereas beehive air and apilarnil are greatly unknown to the patients. Only honey seems to be an acceptable treatment option whereas propolis, pollen and royal jelly seem to be less acceptable. Bee venom was not considered an interesting treatment possibility and, in particular, live bee stings were considered less desirable. This study found that gender and acquaintance with a beekeeper influenced the patients' ratings but age, education and current medical condition did not. CONCLUSION: Live bee stings, apilarnil or the inhalation of beehive air are not appealing to the majority of patients. Before apitherapeutic methods are promoted, it seems to be important to know about patients' willingness to tolerate such treatments. Perhaps therapy modifications can be offered which seem more acceptable.

Similar Characteristics of Endometrial and Endometriotic Epithelial Cells.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial and acquisition of mesenchymal cell characteristics. Our aim was to assess the epithelial phenotype in the pathogenesis of endometriosis with epithelial and mesenchymal markers. We used 2 structural (keratin-18, -19 [K18, K19]), 1 membrane-associated (mucin-1 [MUC1]), and 2 mesenchymal proteins (vimentin; zinc finger E-box-binding homeobox 1, [ZEB1]) to compare epithelial and mesenchymal characteristics in eutopic endometrium with the 3 endometriotic entities, peritoneal, ovarian, and deep infiltrating endometriosis (DIE). Quantitation showed no differences for K18, K19, and MUC1 between endometrium with and without endometriosis. Also, K18 was not different between endometrium and endometriotic lesions. In contrast, K19 and MUC1 were modestly but significantly decreased in the endometriotic lesions compared to endometrium. However, the maintained expression of epithelial markers in all investigated tissues, regardless of the pathological condition, clearly indicates no loss of the epithelial phenotype. This is further supported by the reduced presence of epithelial vimentin in endometriotic lesions which is in contrast to an increase in stromal vimentin in ectopic endometrium, especially in ovarian endometriosis. The ZEB1 increase in endometriotic lesions, especially in DIE, on the other hand suggests a role of partial EMT in the development of endometriotic lesions, possibly connected with the gain of invasive capabilities or stemness. Taken together, although we found some hints for at least a partial EMT, we did not observe a severe loss of the epithelial cell phenotype. Thus, we propose that EMT is not a main factor in the pathogenesis of endometriosis.

Impaired Localization of Claudin-11 in Endometriotic Epithelial Cells Compared to Endometrial Cells.

Claudins are the major components of tight junctions and are often deregulated in human cancer, permitting escape of cancer cells along with the acquisition of invasive properties. Similarly, endometrial cells also show invasive capabilities; however, the role of tight junctions in endometriosis has only rarely been examined. In this study, we analyzed the protein expression and localization of claudin-7 and claudin-11 in human eutopic and ectopic endometrium and endometrial cell lines. We identified claudin-7 primarily at the basolateral junctions of the glandular epithelial cells in eutopic endometrium as well as in the ectopic lesions in nearly all glands and cysts. Quantification of claudin-7 localization by HSCORE showed a slight increase in peritoneal and deep infiltrating endometriosis (DIE) compared to eutopic endometrium. In contrast, claudin-11 was localized mainly in the apicolateral junctions in nearly all glandular epithelial cells of the eutopic endometrium. Interestingly, we observed a deregulation of claudin-11 localization to a basal or basolateral localization in ovarian (P < .001), peritoneal (P < .01), and DIE (P < .05) and a moderately decreased abundance in ovarian endometriosis. In endometrial cell lines, claudin-7 was only present in epithelial Ishikawa cells, and silencing by small-interfering RNA increased cell invasiveness. In contrast, claudin-11 could be demonstrated in Ishikawa and endometriotic 12Z and 49Z cells. Silencing of claudin-11 decreased invasiveness of 12Z slightly but significantly in 49Z. We suggest that although claudin-7 and claudin-11 can be found in nearly all eutopic and ectopic epithelial cells, the impaired localization of claudin-11 in ectopic endometrium might contribute to the pathogenesis of endometriosis.

Composition of the Stroma in the Human Endometrium and Endometriosis.

To analyze whether the endometrial and endometriotic microenvironment is involved in the pathogenesis of endometriosis, we characterized the stromal composition. We used CD90 for fibroblasts, α-smooth muscle actin for myofibroblasts as well as CD10 and CD140b for mesenchymal stromal cells. Quantification of eutopic endometrial stroma of cases without endometriosis showed a high percentage of stromal cells positive for CD140b (80.7%) and CD10 (67.4%), a moderate number of CD90-positive cells (57.9%), and very few α-smooth muscle actin-positive cells (8.5%). These values are highly similar to cases with endometriosis showing only minor changes: CD140b (76.7%), CD10 (63%), CD90 (53.9%), and α-smooth muscle actin (6.9%). There are no significant differences in the composition of CD140b- and CD10-positive stromal cells between the eutopic endometrial stroma and the 3 different endometriotic entities (ovarian, peritoneal, and deep infiltrating endometriosis), except for a significant difference between CD10-positive stromal cells in peritoneal lesions compared to ovarian lesions. However, the percentage of CD90-positive stromal cells was reduced in the 3 different endometriotic entities compared to the endometrium, especially significant in the ovarian lesions. In contrast, the percentage of α-smooth muscle actin-positive cells in the ovary was moderately increased. Taken together, the marker signature of eutopic endometrial and endometriotic stromal cells resembles mostly mesenchymal stromal cells. Our results show clearly that the proportion of the different stromal cell types in the endometrium with or without endometriosis does not differ significantly, thus suggesting that the stromal eutopic endometrial microenvironment does not contribute to the pathogenesis of endometriosis.

Analysis of Clusterin and Clusterin Receptors in the Endometrium and Clusterin Levels in Cervical Mucus of Endometriosis.

Clusterin (CLU) is expressed in tissues and body fluids and is altered in some pathologies. In endometriosis, a noninvasive test is still lacking, thus, we analyzed CLU in mucus samples of patients. Additionally, we investigated localization of CLU and the putative CLU receptors (apolipoprotein E receptor 2 [ApoER2], megalin, very low-density lipoprotein receptor [VLDLR], and transforming growth factor ß receptor type I and II [TßR1/TßR2]). In mucus samples, CLU levels are modestly, but not significantly, higher in cases with endometriosis compared to cases without endometriosis, however, CLU levels are significantly (P = .02) reduced in patients with endometriosis receiving contraception compared to cases with endometriosis without contraception. Analysis of CLU and CLU receptors showed CLU mainly in the uterine epithelial cells in the majority of glands, but also in endothelial cells. Similarly, ApoER2 and TßR1 could also be found preferentially in the endometrial glands. Whereas ApoER2 staining was strong in the vessels, TßR1 was modestly expressed in vessels and muscle cells. In contrast, staining of VLDLR and TßR2 was modest in the glands but stronger in vessels and muscle cells. Megalin staining was faint in the glands. A similar pattern for these proteins could be observed in adenomyosis. We demonstrate for the first time high concentrations of CLU in mucus samples and significantly reduced CLU levels in cases with endometriosis receiving contraception compared to cases with endometriosis without contraception. Furthermore, we identified uterine epithelial and endothelial cells as the main source of CLU and found different preferential CLU receptor complexes on glands, vessels, and smooth muscle cells.

BEMER Therapy Combined with Physiotherapy in Patients with Musculoskeletal Diseases: A Randomised, Controlled Double Blind Follow-Up Pilot Study.

Background. This study evaluates the effect of adjuvant BEMER therapy in patients with knee arthrosis and chronic low back pain in a randomized double blind design. Methods. A total of 50 patients with chronic low back pain and 50 patients with osteoarthritis of knee took part in this study and were randomized into 4 groups. Hospitalized patients received a standardized physiotherapy package for 3 weeks followed by BEMER therapy or placebo. Results. In patients with low back pain, the comparison of the results obtained at the first and second visit showed a significant improvement in resting VAS scores and Fatigue Scale scores. The Oswestry scores and Quality of Life Scale scores showed no change. In patients with knee arthrosis, the comparison of the first and second measurements showed no significant improvement in the abovementioned parameters, while the comparison of the first and third scores revealed a significant improvement in the Fatigue Scale scores and in the vitality test on the Quality of Life Scale. Conclusions. Our study showed that BEMER physical vascular therapy reduced pain and fatigue in the short term in patients with chronic low back pain, while long-term therapy appears to be beneficial in patients with osteoarthritis of knee.

Association of neutrophil extracellular traps with endometriosis-related chronic inflammation.

OBJECTIVE: To study if neutrophil extracellular traps (NETs) are present in the peritoneal fluid of endometriosis patients. NETs play a crucial role in fighting against microorganisms. However, exaggerated NET production may lead to tissue damage in their vicinity in pathological conditions. Our study evaluates the presence of NETs in endometriosis peritoneal fluid. STUDY DESIGN: Peritoneal fluid (PF) was collected in a case-control study from 52 women, who underwent either diagnostic or operative laparoscopy. The control group consisted of 17 women with infertility, chronic pelvic pain, simple or functional cysts or irregular bleeding. The endometriosis group, altogether 35 patients, comprised 19 patients with stage I and II and 16 patients with stage III and IV endometriosis. First we tested whether the PF is able to stimulate NET production. Neutrophils from healthy volunteers were treated with the PF of endometriosis patients and controls and NETs were detected with Sytox orange extracellular DNA dye and immunofluorescence microscopy. Then we evaluated if NETs were already present in the collected PF using the specific myeloperoxidase (MPO)-DNA capture ELISA method, based on the MPO associated with the NET scaffold. RESULTS: The PF of endometriosis patients did not stimulate NET release from healthy granulocytes. However, pre-existent NETs could be detected in 17 endometriosis patients out of 35 (49%). In contrary, in the control group NETs were present in only 3 patients out of 17 (18%), (p=0.03, OR: 4.4). Moreover, the quantification of NETs showed a significantly higher amount of NETs in endometriosis compared to the controls (0.097 vs. 0.02, p=0.04). CONCLUSION: This is the first study, which evaluated and described the presence of NETs in the PF of endometriosis patients. Our study shows, that NETs may be involved in the complex pathophysiology of endometriosis.

[Current treatment of endometriosis with laparoscopic surgery]. / Az endometriosis korszerû laparoszkópos sebészi kezelése.

Surgical treatment of endometriosis aims to remove all visible areas of pelvic endometriosis and restore anatomy by division of adhesions, as well as relieve painful symptoms. In this paper, we summarize the advantages, disadvantages, and efficacy of different laparoscopic surgical procedures in the treatment of endometriosis-associated pelvic pain and infertility. Modern endometrial surgery primarily involves laparoscopy, while indication of previously widespread laparotomy has been restricted to special cases. Surgery for the treatment of peritoneal endometriosis includes several options: electrocoagulation, laser ablation, or excision of the lesions, all of which have similar efficacy in the therapy of endometriosis-associated pelvic pain and infertility. There are two effective techniques for treating ovarian endometrioma: excision (stripping) technique or ablation of the cyst wall. It has been conclusively proven that stripping provides a more favorable outcome than drainage and ablation with regard to alleviating pain symptoms and infertility. The treatment of deeply infiltrating endometriosis involves some of the most challenging dissections in endometrial surgery. Such deeply infiltrating lesions can be most securely removed with laser techniques. For example, rectovaginal septum endometriosis can be completely removed with laser therapy. In case of bowel endometriosis, the affected part of the bowel can be removed by segmental resection, disc resection, or superficial partial-thickness excision. In most cases segmental colorectal resection is employed, because it is the most effective treatment currently available. In case of slight ureteral endometriosis, laparoscopic ureterolysis can be an effective treatment option; however, with obstructive uropathy segmental resection and anastomosis are indicated. Laparoscopic uterosacral nerve ablation and praesacral neurectomy are ancillary procedures meant to further decrease endometriosis-associated pelvic pain symptoms. However, the efficacy of these techniques is not yet proven and currently they appear to offer no added benefits beyond those achievable with conservative surgery alone. The ever improving surgical techniques steadily increase the efficacy of the treatment of endometriosis-associated infertility and pelvic pain, as well as delay recurrence of the disease.

Fetal pathology in second-trimester miscarriages.

OBJECTIVE: We aimed to analyze, based upon autopsy, the main characteristics of miscarriages in the second trimester. METHODS: We have processed the results of fetopathological investigations of 544 aborted fetuses resulting from 486 second-trimester miscarriages. RESULTS: Malformation could be identified in 13.05% of all cases. In almost one third of the patients there was a positive history. In the cases having a malformation, expressed dominance of male fetuses could be observed. Among the fetopathologically identified malformations, 49 were isolated. The most common was the single umbilical artery (22.4%). In 1.3% of the cases a chromosome aberration was verified. CONCLUSION: Miscarriage in pregnancies complicated by a malformation occurs approximately 3 weeks earlier than in cases without a confirmed malformation. There is practically no difference between affected and unaffected miscarriages as far as the cumulative ratio of positive history is concerned. A single umbilical artery alone predisposes to miscarriage, while in association with other malformations it may result from chromosome aberration.

[Common malformations in fetopathologic investigations between 1995 and 2006. Accuracy of ultrasonography confirmed by post mortem investigations]. / Gyakoribb fejlodési rendellenességek a fetopatológiai vizsgálatok anyagában 1995-2006 között. Az ultrahang-diagnosztika hatékonysága a post mortem vizsgálatok tükrében.

UNLABELLED: Fetopathologic investigations are of great importance since they are aimed at assessing the risks of a malformation to recur in a mother's future pregnancy. AIMS: The authors wished to assess and analyse the accuracy of ultrasonography based on the results of fetopathologic investigations in view of malformations of higher prevalence. STUDY DESIGN: The authors have processed the details of 683 cases affecting the nervous, cardiovascular, urinary and skeletal systems, and the abdominal/thoracic walls detected at our department in the period of 1995-2006. RESULTS: No significant differences could be justified as far as the major statistical parameters of maternal and gestational ages at the time of the diagnosis were concerned. There were one or another positive detail in the history in a quarter of malformations affecting the central nervous, cardiovascular and skeletal systems and in one sixth of the cases with disorders of the urinary tract as well as the abdominal/thoracic wall. Urinary tract and cardiovascular malformations were far more common among male fetuses, while moderate female dominance could be observed in malformations of the central nervous system and of the abdominal/thoracic wall. In the four investigated groups of malformations, the proportion of sonographic diagnoses completely coinciding with the post mortem results was found to be approximately or more than 70%, but it was found to be much lower (38%) in urinary malformations. Based on the full sample, the cumulative proportion of coinciding sonographic and fetopathologic diagnoses were more than 63%, while completely incorrect ultrasonographic diagnoses amounted to 18%. CONCLUSIONS: (Even multiple) sonographic investigations are to be performed in a genetic centre if urinary tract malformation with subsequent oligohydramnion is detected. Since the associability of omphalocele and chromosome aberrations has been an established fact, and because some of the cases with omphalocele have been diagnosed as gastroschisis, it may be advisable to perform chromosome investigations in ultrasonographically diagnosed cases of gastroschisis. In cases of VSD, echocardiography should be performed in high-risk pregnancies even if ultrasonography cannot reveal any changes in the patients.

Under-expression of CD24 in pre-eclamptic placental tissues determined by quantitative real-time RT-PCR.

BACKGROUND: Pre-eclampsia is a pregnancy-related disorder present in about 5-7% of all pregnancies. CD24 expression was recently reported in different diseases, while it has not yet been determined in pre-eclamptic placental tissues. METHODS: We collected placental tissues from pre-eclamptic (n = 16) and healthy pregnancies (n = 16). We used the quantitative real-time PCR method with a primer-probe system for determination of CD24 gene expression. RESULTS: We measured CD24 concentrations of 18.94 +/- 26.86 ng/microl in the pre-eclamptic and 53.85 +/- 92.05 ng/microl in the healthy placental tissues (p = 0.03). CONCLUSIONS: The quantitative real-time PCR method is suitable to determine CD24 expression in placental tissues. We suppose the low expression of CD24 may cause the enhanced immune reaction and could play a role in the abnormal development of placenta in pre-eclampsia.

Unilateral triplet ectopic pregnancy after in vitro fertilization and embryo transfer.

OBJECTIVE: To present a case report of a unilateral triplet ectopic pregnancy (EP) conceived by IVF-embryo transfer. DESIGN: Case report. SETTING: University Hospital, Budapest, Hungary. PATIENT(S): A 26-year-old infertile woman with a history of right salpingectomy, hyperprolactinemia, and male factor infertility underwent IVF-embryo transfer of three embryos. Early transvaginal sonography revealed a triplet pregnancy in the left fallopian tube (two at interstitial and one at ampullary location). INTERVENTION(S): Multiple dose methotrexate (MTX) therapy was applied. MAIN OUTCOME MEASURE(S): Follow-up pelvic ultrasounds and laboratory testing confirmed fetal cardiac activity cessation and decreasing beta-hCG levels. RESULT(S): In spite of the decreasing beta-hCG levels the tube's diameter increased, the patient's symptoms escalated, and finally, the level of hemoglobin and hematocrit decreased. Laparotomy was performed with the removal of the left tube and cornual part of the uterus. CONCLUSION(S): Our case represents a very rare condition, a unilateral triplet EP after IVF-embryo transfer-the first one ever reported in the literature. After IVF-embryo transfer early ultrasound examinations are important to identify EPs at an early stage when medical management can still be taken into consideration. Strict monitoring is necessary to identify the success of medical intervention or the need for surgery.

Prenatally diagnosed fetal brain injuries with known antenatal etiologies.

Periventricular leukomalacia of pre- or postnatal onset is responsible for severe neurological and intellectual impairment and cerebral palsy later in life. The etiology is multifactorial, involving hypoxic-ischemic insults of various origin. The disorder is characterized by multiple necrotic foci of the white matter found most frequently adjacent to the lateral ventricles. In the past, intrapartum factors were thought to be the major cause of neonatal brain damage, but recent investigations highlighted the role of antenatal risk factors. We present 4 cases of antenatally diagnosed brain injury with known and unusual etiology.

Effect of hormone replacement therapy on postmenopausal endometrial bleeding.

The aim of the study was to determine the effect of postmenopausal hormone replacement therapy (HRT) (treatment using estrogen only and sequential and continuous combined estrogen-progestogen treatment) on endometrial bleeding and histological changes of the endometrium. In a six-year period (2000-2005), 5893 patients were given care and the incidence of postmenopausal uterine bleeding was detected in groups of patients having and not having received hormonal treatment at the Menopause Outpatient Unit of the authors' department. In the case of bleeding, fractioned abrasion was performed and the samples were analyzed histologically. Among the postmenopausal patients who had not been given hormonal treatment, the incidence of bleeding episodes was significantly higher as among those having received hormonal treatment. In the samples, findings of proliferative endometrium occurred significantly more often in case of non-treated patients and those treated with sequential combined hormone therapy compared to patients receiving continuous combined hormone therapy. Although it was statistically not significant, hyperplasia simplex and complex together showed a tendency of reduced incidence in patients medicated by continuous combined treatment. These findings suggest that continuous combined hormonal treatment started at the right time (even before the menopause) may reduce the chances of the development of hyperplasia. A significantly higher incidence of hyperplasia was noted in patients using estrogen treatment only. It is possible that unopposed estrogen treatment further engraves an already diagnosed endometrial hyperplasia. In the group having received hormonal treatment, no complex hyperplasia accompanied by atypia occurred, only hyperplasia simplex was diagnosed in these cases. As a result of continuous reliance on combined preparations, the endometrium had become atrophied, therefore the chance of hyperplasia-related changes and of bleeding as a side effect decreased significantly. According to the authors' experience, hormonal treatment does not pose a risk to the development of endometrial carcinoma; on the contrary, continuous combined preparations appear to reduce the risk of hyperplasia and, indirectly, the chances of the development of adenocarcinoma.