Positive Ascites Cytology, Postoperative Chemotherapy and Prognosis of Stage I Ovarian Clear Cell Carcinoma.

BACKGROUND/AIM: Ovarian clear cell carcinoma (CCC) is associated with a poor prognosis and is resistant to chemotherapy. The aim of this study was to investigate the prognosis of CCC in Mie prefecture and to identify poor prognostic factors. PATIENTS AND METHODS: In this multi-center retrospective study, we analyzed the data of patients with CCC between February 2012 and December 2020. Patients were staged according to the International Federation of Gynecology and Obstetrics (FIGO) 2014 system. Statistical analyses were performed using the Kaplan-Meier method and compared between the two groups using the log-rank test. RESULTS: A total of 112 patients were included and the median follow-up time was 48 months. There was no difference in the prognosis between stages IA, IC1, and IC2. For patients at stages IA, IC1, and IC2, there was no difference in progression-free survival (PFS) and overall survival between the adjuvant chemotherapy and no chemotherapy groups. Median postrecurrent survival was 18 and 20 months in the stages I-II and III-IV groups, respectively. Multivariate analysis revealed that positive ascites cytology (p=0.006) was associated with PFS for patients at stages I-II and that the stage (p=0.039) was associated with PFS for patients at stages III-IV. CONCLUSION: Positive ascites cytology was a poor prognostic factor for patients at an early stage of CCC. Postoperative chemotherapy could be omitted for patients in stages IA and IC1. Relapsed patients did not respond to the standard treatment and had a poor prognosis regardless of the primary stage.

Evaluation of control of additive concentration in gradient analysis of supercritical fluid chromatography-coupled to tandem mass spectrometry.

Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO2, the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis.

Awareness of Infectious Disease Screening During Early Pregnancy and Knowledge About its Vertical Transmission in Japan: A Report from the Pregnant Women Health Initiative.

OBJECTIVES: We aimed to clarify the accuracy of pregnant women's knowledge and understanding regarding infectious disease screening in early pregnancy and clarify the roles that should be played by health care providers in promoting the health of pregnant women and their children. METHODS: A cross-sectional questionnaire survey was conducted in 25 hospitals across Japan from May 2018 to September 2019. We compared the agreement rates regarding screening results for hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, human T-cell leukemia virus-1 (HTLV-1), and cervical cytology in the medical records and understanding of their results by pregnant women. We then investigated whether participants had knowledge regarding the risk of mother-to child transmission in these diseases and factors associated with their knowledge. RESULTS: We enrolled 2,838 respondents in this study. The rates of agreement for HBV and cervical cancer screening related to human papillomavirus infection were "substantial," those for syphilis was "moderate," and those for HCV and HTLV-1 were "fair," according to the Kappa coefficient. The rate of knowledge regarding mother-to-child transmission of syphilis was highest (37.0%); this rate for the other items was approximately 30%. Increased knowledge was associated with higher educational level and higher annual income. CONCLUSIONS FOR PRACTICE: Pregnant women in Japan had generally good levels of understanding regarding their results in early-pregnancy infectious disease screening. However, they had insufficient knowledge regarding mother-to-child transmission of these diseases. Health care providers should raise awareness in infectious disease prevention among pregnant women and the general public, providing appropriate measures and implementing effective perinatal checkups and follow-ups for infectious diseases.

Plasma citrulline is a sensitive safety biomarker for small intestinal injury in rats.

Plasma citrulline is decreased in cases of severe intestinal injury with apparent villus and cellular atrophy. However, the fluctuation of plasma citrulline in slight intestinal injury remains to be investigated. To clarify this, irinotecan at 30 mg/kg or 60 mg/kg was administered intravenously to rats. Irinotecan reduced plasma citrulline concentrations compared to those in the pair-fed control, being concurrent with slight single cell necrosis and mucosal epithelium regeneration in the small intestine without apparent villus and cellular atrophy. Gene expression of enzymes converting glutamine to citrulline was decreased in the small intestine of the injury model. Moreover, citrulline and arginine levels in the ileum were decreased without alterations to glutamine and glutamate levels, indicating that citrulline synthesis from glutamine was impaired. Metabolome analysis revealed that plasma citrulline and arginine levels were decreased, while there were no marked alterations in other amino acids, metabolites of glycolysis, ketone bodies, or fatty acids. These results suggested that a decreased plasma citrulline level was unlikely to result from amino acid catabolism in response to malnutrition. In conclusion, plasma citrulline concentration reflects slight intestinal injury without apparent villus and cellular atrophy, and thus, it would be a sensitive biomarker for the small intestinal injury.

Pulmonary arterial enlargement predicts cardiopulmonary complications after pulmonary resection for lung cancer: a retrospective cohort study.

BACKGROUND: The finding of pulmonary arterial enlargement on computed tomography has been reported to be associated with pulmonary hypertension. On the other hand, pulmonary hypertension is a known risk factor for thoracic surgery. We investigated whether pulmonary arterial enlargement predicts cardiopulmonary complications following pulmonary resection for lung cancer. METHODS: We reviewed 237 consecutive patients who underwent pulmonary resection for lung cancer. Preoperative patient characteristics (sex, age, Brinkman index, cardiopulmonary comorbidities, cardiothoracic ratio, pulmonary function, and pulmonary arterial enlargement) and surgical data (surgical procedure, pathological stage, postoperative complications, mortality, and length of postoperative hospital stay) were analyzed. In order to evaluate preoperative pulmonary arterial enlargement, we measured the diameter of the main pulmonary artery at its bifurcation and that of the ascending aorta at its widest point using chest computed tomography and calculated the ratio of the former diameter to the latter. RESULTS: In all, 16 patients developed postoperative cardiopulmonary complications and 221 did not. One patient died from postoperative pneumonia. The mean age of patients who developed postoperative cardiopulmonary complications was significantly higher than that of those who did not (78 ± 5 years vs 69 ± 9 years, P=0.0001). The pulmonary artery-to-ascending-aorta ratio was significantly higher in patients who developed postoperative complications than in those who did not (0.94 ± 0.15 vs. 0.81 ± 0.11, P=0.03). Other preoperative patient characteristics and surgical data did not differ significantly between the groups. On multivariate analysis, pulmonary artery-to-ascending-aorta ratio (0.1-point increase; odds ratio 2.3, 95 % confidence interval 1.5-3.5; P=0.0002) and age (1-year increase; odds ratio 1.2, 95 % confidence interval 1.1-1.3; P=0.03) were found to be independent predictors of postoperative cardiopulmonary complications. CONCLUSIONS: A finding of pulmonary arterial enlargement on computed tomography is a potential predictor of postoperative cardiopulmonary complications after lung cancer surgery.

A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA.

5-Hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) represent important epigenetic modifications to DNA, and a sensitive analytical method is required to determine the levels of 5hmC in the genomic DNA of tumor cells or cultured cell lines because 5hmC is present at particular low levels in these cells. We have developed a sensitive liquid chromatography-tandem quadrupole mass spectrometric method for quantifying 5-hydroxymethyldeoxycytidine (5hmdC), 5-methyldeoxycytidine (5mdC), and deoxyguanosine (dG) levels using stable isotope labeled internal standards, and used this method to estimate the global level of 2 modified cytosines in genomic DNA prepared from small number of cells. The quantification limits for 5hmdC, 5mdC and dG were 20pM, 2nM and 10nM, respectively. MRM transitions for isotopologue (isotopologue-MRM) were used to quantify the 5mdC and dG levels because of the abundance of these nucleosides relative to 5hmdC. The use of isotopologue-MRM for the abundant nucleosides could also avoid the saturation of the detector, and allow for all three nucleosides to be analyzed simultaneously without the need for the dilution and re-injection of samples into the instrument. The global ratios of modified cytosine nucleosides to dG were estimated following the quantification of each nucleoside in the hydrolysate of genomic DNA. The limit of estimation for the global 5hmC level was less than 0.001% using 200ng of DNA. Using this method, we found that MLL-TET1, which a fusion protein in acute myelogenous leukemia, did not produce 5hmC, but interfered with TET1 activity to produce 5hmC in cells. Our analytical method is therefore a valuable tool for further studies aiming at a deeper understanding of the role of modified cytosine in the epigenetic regulation of cells.

IDH1 and IDH2 have critical roles in 2-hydroxyglutarate production in D-2-hydroxyglutarate dehydrogenase depleted cells.

D-2-hydroxyglutaric aciduria (D-2HGA) is a hereditary metabolic disorder characterized by the elevated levels of D-2-hydroxyglutaric acid (D-2HG) in urine, plasma and cerebrospinal fluid. About half of the patients have autosomal recessive mutations in D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene. To analyze the origin of D-2HG in D2HGDH-depleted cells, we used small interfering RNA (siRNA) techniques. We found that knockdown of D2HGDH in MCF7 cells increased the levels of 2HG, mimicking D2HGDH mutant cells. Additional knockdown of isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) decreased the level of 2HG in D2HGDH knockdown MCF7 cells. Conversely, ectopic expression of IDH1 or IDH2 increased 2HG in MCF7 cells. These results suggest that IDH1 and IDH2 have roles in production of D-2HG in cells.

Homozygosity of single nucleotide polymorphisms in the 3' region of the canine estrogen receptor 1 gene is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.

Differences in the distribution of single nucleotide polymorphisms (SNPs) and haplotypes in the estrogen receptor α gene (ESR1) were examined in Miniature Dachshunds (n = 48), Chihuahuas (n = 20) and Toy Poodles (n = 18). Five DNA fragments located in the 40-kb region at the 3' end of ESR1 were amplified by polymerase chain reaction and were directly sequenced. We compared allele, genotype and estimated haplotype frequencies at each SNP in the 3' end of ESR1 for these three breeds of small dog. The frequency of the major allele and the genotype frequency of the major allele homozygotes, were significantly higher in Toy Poodles for five SNPs (SNP #5, #14-17) than in Miniature Dachshunds, and significantly higher in Toy Poodles than Chihuahuas for three SNPs (SNP #15-17). A common haplotype block was identified in an approximately 20-kb region encompassing four SNPs (SNPs # 14-17). The frequencies of the most abundant estimated haplotype (GTTG) and GTTG homozygotes were significantly higher in Toy Poodles than in the other two breeds. These results imply that homozygosity for the allele, genotype and haplotype distribution within the block at the 3' end of ESR1 is greater in Toy Poodles than in Miniature Dachshunds and Chihuahuas.

Analysis of single nucleotide polymorphisms in the 3' region of the estrogen receptor 1 gene in normal and cryptorchid Miniature Dachshunds and Chihuahuas.

This study was performed to examine the distribution of single nucleotide polymorphisms (SNPs) and estimated haplotypes in the canine estrogen receptor (ER) alpha gene (ESR1) and the association of them with different phenotypes of cryptorchidism (CO) in Miniature Dachshunds and Chihuahuas. Forty CO and 68 normal dogs were used, and CO was classified into unilateral (UCO; n=33) and bilateral CO (BCO; n=5) or into abdominal (ACO; n=16) and inguinal CO (ICO; n=22). Thirteen DNA fragments located in the 70-kb region at the 3' end of ESR1 were amplified by PCR and sequenced to examine 13 SNPs (#1-#13) reported in a canine SNP database. Ten SNPs (#1-#4, #7, #8, #10-#13) were not polymorphic, and 5 new SNPs (#14-#18) were discovered. A common haplotype block in normal, CO and CO phenotypes was identified for an approximately 20-kb region encompassing 4 SNPs (#14-#17). Allele, genotype and haplotype frequencies in CO without classification by phenotype and also in UCO, ACO and ICO phenotypes were not statistically different from the normal group. Significant differences in genotype frequencies and homozygosity for the estimated GTTG haplotype within the block were observed in BCO compared with the normal group, although the number of BCO animals was small. Our results demonstrate that the examined SNPs and haplotypes in the 3' end of canine ESR1 are not associated with unilateral, abdominal and inguinal CO phenotypes and CO per se in Miniature Dachshunds and Chihuahuas. Further studies are necessary to suggest a clear association between the ESR1 SNPs and bilateral CO in dogs.

Enhancement of prostacyclin synthesis at the beginning of formation of caprine corpora lutea.

We examined changes in the levels of prostacyclin (PGI2) synthase mRNA and 6-keto-PGF1alpha, a stable metabolite of PGI2, in the caprine corpus luteum (CL) during its development and subsequent maintenance. We also looked at the effects of a potent GnRH antagonist (GA), which is known to suppress the release of luteinizing hormone (LH), on the PGI2 synthase mRNA level and the 6-keto-PGF1alpha content during CL development. Goats were divided into a control group (n=12) and a GA-treated group (n=6). They were treated with saline or GA (50 microg/kg, s.c.) on days 0 (day of ovulation), 4, and 8 (control only), and the CL were collected from a subset of goats (n=3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay was performed to quantify the mRNA in the CL using specific cRNA probes generated by RT-PCR and in vitro transcription. The 6-keto-PGF1alpha content in the CL was measured by radioimmunoassay. The level of PGI2 synthase mRNA and the 6-keto-PGF1alpha content in the CL in the control group decreased from day 0 to day 4 (P<0.01), and did not change thereafter from day 4 to day 14. Levels of PGI2 synthase mRNA and 6-keto-PGF1alpha content in the CL on days 4 and 8 were not affected by treatment with GA. These results suggest that PGI2 synthesis is regulated upward at the beginning of caprine CL formation; this may play a role in initiating CL development. This study also suggests that changes of PGI2 synthesis during CL development are probably not regulated by LH.

Changes of messenger RNAs encoding vascular endothelial growth factor and its receptors during the development and maintenance of caprine corpora lutea.

The present study was conducted to examine changes of mRNAs encoding vascular endothelial growth factor (VEGF) and its receptors (KDR/Flk-1 and Flt-1), and CD34, which is known to be a specific marker for endothelial cells, during the development and maintenance of the caprine corpora lutea (CL). Effects of a potent GnRH antagonist (GA), which was previously shown to suppress release of luteinizing hormone (LH), on expressions of those mRNAs during the CL development were also investigated. Goats were divided into control (n = 12) and GA-treated groups (n = 6). The goats were treated with saline or GA (50 microg/kg, sc) on days 0 (day of ovulation), 4, and 8 (control only), and CL collected on a subset of goats (n = 3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay was performed to quantitate the mRNAs in the CL using specific cRNA probes generated by RT-PCR and in vitro transcription. Level of CD34 mRNA significantly increased from day 0 to 8 (CL development) in the control group (P < 0.05). Long and short forms were detected in the caprine CL by RT-PCR for VEGF mRNA and analyses of their sequences showed that they correspond to mRNAs encoding VEGF(165) and VEGF(121), respectively. Level of VEGF(165) mRNA significantly increased from day 4 to 8 and day 8 to 14 (CL maintenance) in the control group (P < 0.05) while VEGF(121) mRNA did not change during the whole period. Level of KDR/Flk-1 mRNA significantly increased from day 0 to 8 (P < 0.05) while Flt-1 mRNA significantly increased from day 8 to 14 (P < 0.005) in the control group. In the GA-treated group, levels of all of the mRNAs did not alter remarkably as compared with those in the control group. These results suggest that rise of KDR/Flk-1 and VEGF(165) mRNAs during the caprine CL development may be associated with enhanced angiogenesis and that increment of VEGF(165) and Flt-1 mRNAs during the CL maintenance may play nonangiogenic roles. The present study also indicates that the changes of VEGF(165) and KDR/Flk-1 mRNAs during the CL development are probably not regulated by LH.

Inhibition of luteinizing hormone receptor expression during the development of caprine corpora lutea by administration of gonadotropin-releasing hormone antagonist.

The present study was conducted to examine effects of a potent GnRH antagonist (GA), which suppresses release of luteinizing hormone (LH), on LH receptor expression during the development of the caprine corpus luteum (CL). Goats were divided into control and GA-treated groups. The goats were treated with saline or GA (50 microg/kg, s.c.) on days 0 (day of ovulation), 4 and 8 (control only), and CL collected on a subset of goats (n = 3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay and [(125)I]-hCG binding assay were performed to quantitate mRNA and protein of the LH receptor in the CL, respectively. On day 4, CL weight, levels of LH receptor mRNA and protein in the GA-treated group were similar to those of the control group. By day 8, CL weight and levels of LH receptor mRNA and protein in the GA-treated group were reduced relative to those of the control group (P < 0.05). There was no difference of affinity of the LH receptor between both groups on day 8. These results suggest that the treatment with GA inhibits gene and protein expressions of the LH receptor during the development of CL in the goat, and thus, support an idea that endogenous LH participates in the increase of its own receptor.