FLASH Effect Is Not Always Induced by Ultra-high Dose-rate Proton Irradiation Under Hypoxic Conditions.

BACKGROUND/AIM: Pre-clinical studies have shown that irradiation with electrons at an ultra-high dose-rate (FLASH) spares normal tissue while maintaining tumor control. However, most in vitro experiments with protons have been conducted using a non-clinical irradiation system in normoxia alone. This study evaluated the biological response of non-tumor and tumor cells at different oxygen concentrations irradiated with ultra-high dose-rate protons using a clinical system and compared it with the conventional dose rate (CONV). MATERIALS AND METHODS: Non-tumor cells (V79) and tumor cells (U-251 and A549) were irradiated with 230 MeV protons at a dose rate of >50 Gy/s or 0.1 Gy/s under normoxic or hypoxic (<2%) conditions. The surviving fraction was analyzed using a clonogenic cell survival assay. RESULTS: No significant difference in the survival of non-tumor or tumor cells irradiated with FLASH was observed under normoxia or hypoxia compared to the CONV. CONCLUSION: Proton irradiation at a dose rate above 40 Gy/s, the FLASH dose rate, did not induce a sparing effect on either non-tumor or tumor cells under the conditions examined. Further studies are required on the influence of various factors on cell survival after FLASH irradiation.

Differentiation of ovarian serous carcinoma from ovarian clear cell carcinoma using a 10-gene signature selected by comprehensive gene expression analysis.

AIM: Ovarian serous carcinoma (OSC) and ovarian clear cell carcinoma (OCCC) are two major histological types of epithelial ovarian carcinoma (EOC), each with different biological features and clinical behaviors. Although immunostaining is commonly used for differential diagnosis between OSC and OCCC, correct identification of EOC with mixed-type histology is sometimes a diagnostic challenge. The aim of the present study was to explore candidate genes as potential diagnostic biomarkers that distinguish OSC from OCCC. METHODS: A total of 57 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery. Total RNAs were extracted from fresh-frozen tissues of EOC patients, and were used for comprehensive gene expression analysis using DNA microarray technology. RESULTS: Ten candidate genes, FXYD2, TMEM101, GABARAPL1, ARG2, GLRX, RBPMS, GDF15, PPP1R3B, TOB1, and GSTM3 were up-regulated in OCCC compared to OSC. All EOC patients were divided into two groups according to hierarchical clustering using a 10-gene signature. CONCLUSION: Our data suggest that the 10 candidate genes would be an excellent marker for distinguishing OSC from OCCC. Furthermore, the molecular signatures of the 10 genes may enlighten us on the differences in carcinogenesis, and provide a theoretical basis for OCCC's resistance to chemotherapy in the future.

Successful management of pelvic recurrence of MSI-High endometrial cancer by total pelvic exenteration followed by administration of pembrolizumab:A case report.

Surgery can be curative treatment for pelvic locoregional recurrence of endometrial cancer; however, a cure is contingent on complete resection. Here, we report the case of a patient in whom recurrent endometrial tumor remained in the pelvis after resection; long-term control was achieved with postoperative administration of pembrolizumab.The patient had recurrent endometrial cancer of stage IA and was treated with chemotherapy and radiation, but tumor persisted in the pelvic cavity. We therefore attempted total pelvic exenteration, but the tumor was adherent to the pelvic wall and complete resection could not be achieved. However, postoperative administration of pembrolizumab controlled the residual tumor for more than two years without regrowth. We believe that since the resected tumor was MSI-High, the residual tumor responded well to pembrolizumab. It is not known whether cytoreductive surgery contributes to a long-term response to pembrolizumab, but at least in our patient, pembrolizumab appeared to be a very effective drug therapy for MSI-High endometrial cancer that was refractory to chemotherapy and radiotherapy.

Effect of gestational weight gain on preeclampsia among underweight women: A single tertiary referral center study in Japanese women.

AIM: To examine the effect of weight gain during pregnancy on preeclampsia among women with a prepregnancy body mass index < 18.5 kg/m2 . METHODS: This retrospective cohort study included 479 Japanese women with singleton pregnancies and a prepregnancy body mass index < 18.5 kg/m2 , who gave birth between 2013 and 2019 at Ohta Nishinouchi Hospital. The study included 22 (18 with preeclampsia and four with gestational hypertension) and 457 patients with and without hypertensive disorders of pregnancy, respectively. RESULTS: The prevalence of hypertensive disorders of pregnancy and preeclampsia was 4.6% and 3.8%, respectively. With weight gain during pregnancy (continuous variable) set as a reference, multiple logistic regression revealed that excessive weight gain during pregnancy increased the risk of preeclampsia (adjusted odds ratio: 1.13, 95% confidence interval: 1.00-1.28, p < 0.05) and hypertensive disorders of pregnancy (adjusted odds ratio: 1.15, 95% confidence interval: 1.03-1.29, p < 0.05). Based on receiver operating characteristic curve analyses (area under the curve 0.65, 95% confidence interval: 0.50-0.80; p < 0.05), we determined the cutoff value of weight gain during pregnancy for the occurrence of preeclampsia among women with body mass index < 18.5 kg/m2 to be 13.0 kg, with sensitivity and specificity of 0.50 and 0.78, respectively. CONCLUSION: This study indicates that excessive weight gain during pregnancy increases preeclampsia risk among underweight women and provides new recommendations for weight gain during pregnancy for such women. Further research regarding the pathogenesis of preeclampsia for underweight women is warranted.

Association of placenta previa with placental size in Japanese women: A study from a single tertiary referral center.

AIM: The effect of placenta previa on age-specific placental size has not yet been elucidated. This study aimed to examine the effect of placenta previa on the Japanese standardized z-scores of placental size. METHODS: This retrospective cohort study included Japanese participants from Ohta Nishinouchi Hospital with single pregnancies who gave birth during 2013-2019. The participants were categorized into two groups based on the presence or absence of placenta previa. Multiple linear regression analyses were used to identify the association of placenta previa with the z-score of placental size, after adjusting for factors, such as maternal smoking status, maternal age, assisted reproductive technology, myoma uteri, uterine anomaly, maternal hypertension at the time of pregnancy, and body mass index before pregnancy. RESULTS: A total of 4071 Japanese women (76 with placenta previa and 3995 without placenta previa) were identified. Placenta previa significantly increased the placental weight z-score (partial regression coefficient: 0.44, 95% confidence interval 0.10-0.70, p < 0.001). CONCLUSION: Placenta previa increased the age-specific placental size. Further studies are required to examine whether placenta previa is associated with the risk of obstetrics complications related to the placental size.

Identification and clinical significance of somatic oncogenic mutations in epithelial ovarian cancer.

OBJECTIVE: Epithelial ovarian cancer (EOC) is a heterogeneous disease with diverse clinicopathological features and behaviors, and its heterogeneity may be concerned with the accumulation of multiple somatic oncogenic mutations. The major goals of this study are to systematically perform the comprehensive mutational profiling in EOC patients, and investigate the associations between somatic mutations and clinicopathological characteristics. METHODS: A total of 80 surgical specimens were obtained from EOC patients who had previously undergone primary debulking surgery, and genomic DNAs were extracted from fresh-frozen tissues. We investigated mutational status in hot spot regions of 50 cancer-related genes by targeted next-generation sequencing using an Ion AmpliSeq Cancer Hotspot Panel v2 Kit. RESULTS: Validated mutations were detected in 66 of the 80 tumors (82.5%). The five most frequently mutated genes were TP53 (43.8%), PIK3CA (27.5%), KRAS (23.8%), PTEN (10%) and CTNNB1 (10%). PTEN and CTNNB1 mutations were associated with younger age. PIK3CA1, KRAS and CTNNB1 mutations were observed in early-stage, whereas TP53 mutations were more common in advanced stage. Significant associations were observed between TP53 mutation and serous carcinoma, and between KRAS mutation and mucinous carcinoma. Both PIK3CA mutation and CTNNB1 mutation were also significantly associated with endometrioid and clear cell carcinoma. The patients with PIK3CA and KRAS mutations were significantly associated with favorable progression free survival (PFS). In particular, PIK3CA mutations had more significant associations with favorable PFS than PIK3CA wild-type in the endometrioid subtype (P = 0.012). Patients with mutations only in TP53 were significantly associated with worse PFS. CONCLUSION: EOCs were heterogeneous at the genomic level and harbored somatic oncogenic mutations. Our molecular profiling may have the potential for becoming a novel stratification within histological subtypes of EOC. Further studies are needed to define molecular classification for improved clinical outcomes and treatment of EOC patients in future.

First trimester heterotopic pregnancy with shock treated laparoscopically, followed by uneventful term pregnancy and normal birth.

Heterotopic pregnancy (HP), a coexistence of intrauterine and ectopic pregnancies, is extremely rare. Although there have been many reports of maternal outcomes in pregnant women with HP, they have not described fetal neurodevelopmental outcomes and survival. A 30-year-old Japanese woman in early gestation who had undergone two previous cesarean deliveries was transferred to our hospital with vital signs of shock. HP was confirmed by ultrasonography and laparoscopic surgery, and right salpingectomy was performed. At term, a 2,875 g neonate was delivered via cesarean section without any complications.

Effect of gestational weight gain on placental weight: A single tertiary referral center study of Japanese women.

AIM: The effect of gestational weight gain on placental weight has not been elucidated. We aimed to examine the effect of body weight gain during pregnancy on the Japanese standardized z-score of placental weight, based on the pre-pregnancy body mass index. METHODS: This retrospective cohort study included Japanese women with singleton pregnancies who gave birth during 2013-2019 at Ohta Nishinouchi Hospital. Participants (n = 3610) were categorized by their pre-pregnancy body mass index: G1 (<18.5 kg/m2 ), G2 (18.5 to <20.0 kg/m2 ), G3 (20.0 to <23.0 kg/m2 ), G4 (23.0 to <25.0 kg/m2 ), and G5 (≥25.0 kg/m2 ). Multiple linear regression analysis was used to identify associations between insufficient or excessive gestational weight gain in each body mass index category and z-score of placental weight, with adjustments for maternal age, assisted reproductive technology, and maternal pre-pregnancy conditions, such as hypertension, diabetes mellitus, myoma uteri, and uterine anomalies. RESULTS: Among the 3610 women assessed, 479, 692, 1292, 435, and 711 were in G1-G5, respectively. In G1, G3, and G4, excessive weight gain increased the placental weight z-score ([B: 0.50, 95% confidence interval [CI]: 0.23-0.76], [B: 0.19, 95% CI: 0.19-0.33], and [B: 0.18, 95% CI: 0.10-0.26]). Insufficient weight gain decreased the placental weight z-score in G3 (B: -0.19, 95% CI: -0.33 to -0.06) and G4 (B: -0.21, 95% CI: -0.29 to -0.13) women. CONCLUSION: The effect of weight gain during pregnancy on placental size varies by pre-pregnancy body mass index. This result may guide personalized pre-conception counseling to improve the outcomes of offspring.

Unexpected placenta accreta spectrum after the use of assisted reproductive technology in women with adenomyomectomy.

Placenta accreta spectrum (PAS) is a rare complication that can lead to life-threatening postpartum hemorrhage. PAS can sometimes occur unexpectedly, without placenta previa;such cases can lead to higher maternal mortality and morbidity than expected cases. Here, the authors report a case of unexpected PAS caused by assisted reproductive technology (ART) in a woman with adenomyosis. The patient was a 37-year-old Japanese primipara woman who presented to our hospital at 11 weeks gestation, later returning to her parents' house to give birth. The woman had adenomyosis and underwent adenomyomectomy, which was followed by an ART pregnancy. The patient was admitted to our hospital because of a life-threatening preterm birth, with a short cervix and no evidence of placenta previa. Despite strict perinatal management, preterm rupture of the membrane (PROM) occurred. During laparotomy, the small intestine, rectum, and both right and left ovaries were clumped together and severely adhered to the surface of the uterus. After delivery, manual partial removal of the placenta was performed, resulting in heavy bleeding from the implantation site, which was diagnosed as an unexpected PAS. Following several uterine compression efforts, we successfully preserved the uterus.

Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma.

PURPOSE: Endometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients. METHODS: A total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes. RESULTS: Validated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion. CONCLUSION: Our comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.

Clinical implication of oncogenic somatic mutations in early-stage cervical cancer with radical hysterectomy.

It is well known that tumour initiation and progression are primarily an accumulation of genetic mutations. The mutation status of a tumour may predict prognosis and enable better selection of targeted therapies. In the current study, we analysed a total of 55 surgical tumours from stage IB-IIB cervical cancer (CC) patients who had undergone radical hysterectomy including pelvic lymphadenectomy, using a cancer panel covering 50 highly mutated tumorigenesis-related genes. In 35 patients (63.6%), a total 52 mutations were detected (58.3% in squamous cell carcinoma, 73.7% in adenocarcinoma), mostly in PIK3CA (34.5%) and KRAS and TP53 (9.1%). Being mutation-positive was significantly correlated with pelvic lymph node (PLN) metastasis (P = 0.035) and tended to have a worse overall survival (P = 0.076). In particular, in the patients with squamous cell carcinoma, there was a significant association between being mutation-positive and relapse-free survival (P = 0.041). The patients with PLN metastasis had a significantly worse overall survival than those without (P = 0.006). These results indicate that somatic mutation status is a predictive biomarker for PLN metastasis in early-stage CC, and is consequently related to poor prognosis. Therefore, comprehensive genetic mutations, rather than a single genetic mutation, should be examined widely in order to identify novel genetic indicators with clinical usefulness.

Successful Management of Platinum-resistant Ovarian Cancer by Weekly Nedaplatin Followed by Olaparib: Three Case Reports.

BACKGROUND: Treatment for platinum-resistant ovarian cancer is difficult and challenging because available chemotherapeutic agents only offer short survival improvements. The efficacy of re-treatment with platinum-based agents including nedaplatin for platinum-resistant patients has not been fully investigated. CASE REPORT: We describe herein three cases of heavily treated platinum-resistant ovarian cancer that were successfully treated with weekly nedaplatin followed by olaparib. After becoming platinum-resistant, the cases were treated with non-platinum chemotherapies. Following these regimens, weekly nedaplatin was introduced, followed by olaparib. At the time of writing, survival since the start of weekly nedaplatin was 30 months for case 1, 20 months for case 2, and 17 months for case 3, with all patients showing no evidence of disease. CONCLUSION: Weekly nedaplatin followed by olaparib might represent a good treatment option for platinum-resistant ovarian cancer and is a solid candidate for further evaluation.

Successful management of preoperatively diagnosed torsion of a subserosal uterine fibroid by pneumoperitoneum laparoscopic single-port surgery.

OBJECTIVE: Preoperative diagnosis and successful management of acute torsion of a subserosal fibroid by using appropriate imaging modalities and single-port laparoscopic surgery. CASE REPORT: A 44-year-old nulliparous woman presented with lower abdominal pain. Computed tomography and magnetic resonance imaging with contrast enhancement revealed a tumor in the pouch of Douglas with a low contrast at the center and thin-rim enhancement. Torsion of a uterine subserosal fibroid was diagnosed preoperatively. Laparoscopic single-port surgery by pneumoperitoneum was performed. Torsion of the pedicle attached to the uterine wall was excised by bipolar coagulation and cut with scissors. The extirpated fibroid was extracted from the umbilical wound. The pneumoperitoneum single-port laparoscopic surgery was completed as a gynecologic emergency operation. CONCLUSION: Torsional uterine fibroids are difficult to diagnose preoperatively as symptoms are nonspecific and need emergent surgical management as an acute abdomen. Preoperative diagnosis using appropriate imaging modalities is important to perform single-port laparoscopic surgery.

Prediction of lymphovascular space invasion in endometrial cancer using the 55-gene signature selected by DNA microarray analysis.

Lymphovascular space invasion (LVSI) is considered to be the beginning of lymphogenous and hematogenous metastases. It is strongly related to dissemination, and therefore could be a valuable predictive sign of lymph node metastases and distant spread. Recently, the presence of LVSI in endometrial cancer (EC) has been shown to be an independent prognostic factor. The preoperative diagnosis of LVSI by pathological examination is difficult and LVSI is detected after surgery. The aim of the current study was to explore candidate genes as potential diagnostic biomarkers and determine whether they are predictors of LVSI in patients with EC. A total of 88 surgical specimens obtained from EC patients who had undergone surgical resection at Fukushima Medical University Hospital between 2010 and 2015 were analyzed using DNA microarray. LVSI was significantly associated with poor prognostic factors in EC such as higher tumor grade, lymph node metastasis, deep myometrium invasion, advanced stage and recurrence. Fifty-five candidate genes were significantly differentially expressed between 26 LVSI-positive and 62 LVSI-negative samples. All 88 samples were divided into two groups according to hierarchical clustering of 55 genes. Regarding diagnostic accuracy, sensitivity and negative predictive value were both high (92% and 95%, respectively); further, specificity and positive predictive value were both moderate (63% and 71%, respectively). Our data suggests that the 55-gene signature could contribute to predicting LVSI in EC, and provide clinically important information for better management. The molecular signatures of 55 genes may be also useful for understanding the underlying mechanism of LVSI.

Unique Learning System for Uterine Artery Embolization for Symptomatic Myoma and Adenomyosis for Obstetrician-Gynecologists in Cooperation with Interventional Radiologists: Evaluation of UAE From the Point of View of Gynecologists Who Perform UAE.

STUDY OBJECTIVE: To evaluate a unique learning system for uterine artery embolization (UAE) and examine its feasibility and clinical outcomes for the treatment of symptomatic uterine leiomyomas and adenomyosis when performed by obstetrician-gynecologists in cooperation with interventional radiologists (IVRs). DESIGN: Retrospective study (Canadian Task Force classification II-2). SETTING: University hospital. PATIENTS: One hundred seventy-three patients who underwent UAE for symptomatic leiomyomas and adenomyosis. INTERVENTIONS: We examined the medical records of patients who underwent UAE for symptomatic uterine leiomyomas and adenomyosis at our department between 2003 and 2012 using our learning system for UAE for obstetrician-gynecologists in cooperation with IVRs. The charts of all patients were reviewed, and data on etiologic factors, past medical history of leiomyomas and adenomyosis, symptoms, details of UAE, and clinical outcomes after UAE were extracted. MEASUREMENTS AND MAIN RESULTS: A total of 173 patients who underwent 177 UAEs were identified, including 4 patients who underwent embolization twice because of primary treatment failure or symptom recurrence. During the study period, 2 gynecologists successfully acquired endovascular skills. The technical success rate was 97.7% (174 of 177). The duration of fluoroscopy in procedures performed by obstetrician-gynecologists who acquired endovascular skills was not significantly different from that in procedures performed by IVRs at our institution; however, this duration was significantly longer in procedures performed by obstetrician-gynecologists who did not have sufficient experience with our learning protocol for UAE because of inadequate live observation of UAEs performed by skilled IVRs. Complications that necessitated discontinuation of the procedure occurred in 2.3% of cases (4 of 177). The clinical outcomes were similar to those reported in previous studies. Adverse events after UAE included myeloid passages in 7.0% (11 of 158), infections in 2.5% (4 of 158), vaginal discharge in 2.5% of patients with leiomyomas (4 of 158), and vaginal discharge in 7.1% of patients with adenomyosis (1 of 14). All the adverse events were adequately treated by the obstetrician-gynecologists themselves. The timing of hysterectomy due to complications or recurrence of symptoms after UAE varied widely. CONCLUSION: UAE performed by obstetrician-gynecologists in cooperation with radiologists can be achieved safely and successfully with acceptable clinical outcomes. Live observation of the procedure performed by skilled IVRs is essential to improving the skills and reducing the fluoroscopic time of obstetrician-gynecologists.

Dissociation of biomolecules in liquid environments during fast heavy-ion irradiation.

The effect of aqueous environment on fast heavy-ion radiation damage of biomolecules was studied by comparative experiments using liquid- and gas-phase amino acid targets. Three types of amino acids with different chemical structures were used: glycine, proline, and hydroxyproline. Ion-induced reaction products were analyzed by time-of-flight secondary-ion mass spectrometry. The results showed that fragments from the amino acids resulting from the C-Cα bond cleavage were the major products for both types of targets. For liquid-phase targets, specific products originating from chemical reactions in solutions were observed. Interestingly, multiple dissociated atomic fragments were negligible for the liquid-phase targets. We found that the ratio of multifragment to total fragment ion yields was approximately half of that for gas-phase targets. This finding agreed with the results of other studies on biomolecular cluster targets. It is concluded that the suppression of molecular multifragmentation is caused by the energy dispersion to numerous water molecules surrounding the biomolecular solutes.

Significance of immature platelet fraction and CD41-positive cells at birth in early onset neonatal thrombocytopenia.

Early thrombocytopenia is a common hematological abnormality in sick neonates. Here, we examined the relationship between early thrombocytopenia in neonates and parameters associated with thrombopoiesis to identify predictive factors at birth. Two hundred and forty-four neonates admitted to the neonatal intensive care unit were divided into thrombocytopenic (n = 55, 23%) and non-thrombocytopenic (n = 189, 77%) groups based on platelet counts, which were monitored within 72 h of birth. Immature platelet fraction (IPF) and platelet count at birth were determined simultaneously soon after phlebotomy with an automated hematology analyzer. Megakaryocytes and their precursors positive for CD41 in peripheral blood were examined at birth by flow cytometry. The thrombocytopenic group showed significantly higher IPF percentage and lower percentage of CD41+ mononuclear cells (MNCs) than did the non-thrombocytopenic group (P < 0.01). Moreover, the percentage of CD41+ MNCs significantly differentiated neonates with platelet counts >150 x 10(3)/microL at birth and nadir platelet count <150 x 10(3)/microL over the clinical course from neonates without thrombocytopenia. These observations suggest that the percentage of CD41+ MNCs at birth and IPF percentage are useful predictors of early thrombocytopenia in the majority of sick neonates.

Crystal structures and topochemical polymerizations of 7,7,8,8-tetrakis(alkoxycarbonyl)quinodimethanes.

Highly conjugated monomers, 7,7,8,8-tetrakis(alkoxycarbonyl)quinodimethanes (methoxy (1a), ethoxy (1b), isopropoxy (1c), benzyloxy (1d), chloroethoxy (1e), and bromoethoxy (1f)), were synthesized. Recrystallizations of 1a, 1c, 1e, and 1f yielded two crystal forms (prisms (1a-A) and needles (1a-B), needles (1c-A) and plates (1c-B), prisms (1e-A) and plates (1e-B), and prisms (1f-A) and needles (1f-B)), which have different molecular packing modes by X-ray crystal structure analysis, indicating that the crystals are polymorphic. In the photopolymerizations of these monomer crystals in the solid state, 1a-A, 1e-A, and 1f-A polymerized topochemically to give crystalline polymers. For their thermal polymerizations in the solid state, in addition to 1a-A, 1e-A, and 1f-A, 1e-B and 1f-B polymerized, but polymers formed from the 1e-B and 1f-B were amorphous. The packing of quinodimethane molecules in the crystals was defined by four kinds of parameters, stacking distance (d(s)), the distance between the reacting exomethylene carbon atoms (d(cc)), the angles formed between the stacking axis and longer axis of the monomer molecule (theta(1)), and the shorter axis of the monomer molecule (theta(2)), and then the polymerization reactivity of these quinodimethanes in the solid state was discussed on the basis of these parameters.