Genetic Profile and Microsatellite Instability in a Case of Secondary Esophageal Squamous Cell Carcinoma 12 Years After Allogeneic Hematopoietic Stem Cell Transplantation for Aplastic Anemia.

We report on a 16-year-old Japanese boy in whom an esophageal squamous cell carcinoma (ESCC) developed 12 years after allogeneic hematopoietic stem cell transplantation was performed for aplastic anemia. A high frequency of microsatellite instability was detected in samples of ESCC. Moreover, the detection of pathogenic variants, including single nucleotide substitution of TP53 (c.346C>T) and BRCA2 (c.6952C>T) and splicing of KDM6A (c.1194+2T>G), suggest that the development of ESCC in the patient was triggered by impairment of checkpoint and repair for DNA damage and epigenetic modification through accumulation of gene mutations induced by chronic graft-versus-host disease and prolonged administration of tacrolimus.

Classification of "Kintoki ninjin" and other groups of carrot (Daucus carota) based on simple sequence repeat markers.

Carrot (Daucus carota) is cultivated in temperate regions for its taproot. Eastern and Western types have been differentiated. In Japan, the former type is categorized into Kintoki, Takinogawa oonaga, and Toso, with a few local cultivars. However, their genetic relationships are unclear because of the paucity of reports. We classified the Japanese Eastern and selected Western types based on simple sequence repeat (SSR) markers. Field traits, including root weight, length, diameter, and skin color, were also examined. Our field tests showed clear differences between the Kintoki and Western-type cultivars, confirming their differentiation. A phylogram based on nine SSRs classified 24 cultivars into groups I and II. Group I included all Eastern-type carrots examined (Kintoki and Toso groups, plus two local and two foreign cultivars), with the exception of an Indian cultivar ('Pusa rudhira red'). Among them, red carrots including Kintoki were clustered into two subgroups. Western-type, Eastern-Western hybrid, and 'Pusa rudhira red' were included in group II. A population structure analysis revealed the split between the Eastern and the other types. This study elucidates the genetic characteristics of the Eastern type of carrot, which will be valuable information for carrot breeding, especially when using the Eastern type as a source.

Characteristic differences and reference ranges for mitral, tricuspid, aortic, and pulmonary Doppler velocity waveforms during fetal life.

OBJECTIVES: We aimed to construct reference ranges for time intervals of each component of cardiac flow velocity waveforms in normal fetuses, comparing those variables between right and left ventricles. METHODS: In 359 fetuses at the gestational age of 17-38 weeks, the durations of atrioventricular (AV) valve opening (AVVO), AV valve closure (AVVC), total E- (total-E) and A- (total-A) waves, total ejection time (total-ET), acceleration time (acc-E for E-wave, acc-A for A-wave, and acc-ET for ejection time), and deceleration time (dec-E for E-wave, dec-A for A-wave, and dec-ET for ejection time) were studied cross sectionally. RESULTS: Both right and left acc-E showed the strongest correlations with gestational age (r = 0.478 and r = 0.519, respectively). Left AVVO showed a stronger correlation (r = 0.474) than right AVVO (r = 0.282) and, conversely, right AVVC showed a stronger correlation (r = 0.399) than left AVVC (r = 0.195) with gestational age. Significant differences (all P values <0.001) were observed for all right and left parameters other than total-A and acc-E. CONCLUSIONS: Characteristic differences between right and left ventricles were found in the reference ranges, suggesting the developmental properties of the fetal heart. © 2014 John Wiley & Sons, Ltd.

[Analysis of molecular mechanism involved in development of acute myeloid leukemia].

We examined the role of molecules related to drug resistance, such as P-glycoprotein (P-gp) and telomerase (TERT), signaling molecules of STATs and FLT3 in leukemia pathogenesis in de novo acute myeloid leukemia (AML), and myelodysplastic syndrome in the phase of overt leukemia (MDS-OL). Subjects were 18 patients with de novo AML, in which expression of P-gp, TERT, STAT3, STAT5, and FLT3 was observed in 11, 14, 16, 18, and 14 of patients, respectively. Phosphorylation of STAT3, STAT5, and FLT3 in patients with de novo AML was observed in 10 out of 14, 14 out of 18, and 10 out of 14 patients, respectively. Phosphorylation of STAT5 was associated with expression of both P-gp and TERT, suggesting that STAT5 is one of the transcription factors for these genes. On the other hand, P-gp, TERT, STAT3, STAT5, and FLT3 were expressed in 3, 1, 1, 6, and 1 of the 7 patients with MDS-OL, respectively. While phosphorylation of STAT5 was observed in 4 out of 7 patients, phosphorylation of STAT3 or FLT3 was not detected in all cases examined. Telomere length varied from 2.7 kb to 6.0 kb in de novo AML, accompanied by an increased level of telomerase activity in 4 of 5 patients with de novo AML. In contrast, all MDS-OL cases showed a similar telomere length of 4-5 kb. These results indicate that consideration should be given to the differences of molecular mechanisms in the pathogenesis of de novo AML and MDS-OL for the treatment strategy of AML.

NF-κB signaling activation via increases in BRD2 and BRD4 confers resistance to the bromodomain inhibitor I-BET151 in U937 cells.

Novel epigenetic therapies targeting bromodomain and extra-terminal (BET) family proteins have shown therapeutic efficacy in diverse hematologic malignancies and solid cancers. However, the mechanism of resistance remains poorly understood. In the present study, we evaluated the mechanism of resistance to the BET inhibitor I-BET151 and its signaling pathway to overcome resistance in U937 cells. Treatment with 10 µM I-BET151 significantly induced growth inhibition, apoptosis, and cell cycle modulation, including increases in sub-G1 and G1 phases and decreases in S and G2/M phases, in U937 cells. However, no significant changes in these factors were detected in I-BET151-resistant U937 (U937R) cells. Combined treatment with I-BET151 and IKK inhibitor VII synergistically induced apoptosis in U937 and U937R cells. Increased expression of bromodomain-containing protein (BRD) 2, BRD4, and nuclear NF-κBp65 proteins was detected in U937R cells. IKK inhibitor VII inhibited the activation of NF-κBp65 protein in the nuclear fraction of U937R cells. These findings suggest that resistance to I-BET151 in U937R cells is related to constitutive activation of the NF-κB signaling pathway via increased expression of both BRD2 and BRD4. Targeting the NF-κB signaling pathway may be an effective therapeutic strategy to enhance or restore the sensitivity to I-BET151 in U937 cells.

Dandelion T-1 extract up-regulates reproductive hormone receptor expression in mice.

Reproductive hormones exert their actions via receptors in diverse tissues. In this study, we examined the expression of estrogen receptors (ERalpha and ERbeta), progesterone receptor (PR), and follicle-stimulating hormone receptor (FSHR) in the adipose tissue and reproductive organs of mice following oral treatment with Dandelion T-1 extract (DT-1E) for 6 weeks. Quantitative assays by real-time reverse transcription polymerase chain reaction showed enhanced expression of ERalpha and ERbeta mRNA in the adipose tissue of mice fed a diet containing DT-1E compared with the control group fed a plain diet. Furthermore, following gonadotropin injection, higher mRNA expression of ERalpha, ERbeta and PR in the uterus and FSHR in the ovary was found in the DT-1E group compared with the control group. An immunohistochemical study also showed increased levels of the above-mentioned receptors in the DT-1E group. The present study shows that oral intake of DT-1E up-regulates ERalpha, ERbeta, PR and FSHR expression in mice, suggesting the potential application of DT-1E for the clinical treatment of reproductive hormone-related disturbances.

Intractable bone marrow edema syndrome of the hip.

There is a need for an effective and noninvasive treatment for intractable bone marrow edema syndrome of the hip. Forty-six patients with intractable bone marrow edema syndrome of the hip were retrospectively studied to compare the short-term clinical effects of treatment with high-energy extracorporeal shock wave therapy vs femoral head core decompression. The postoperative visual analog scale score decreased significantly more in the extracorporeal shock wave therapy group compared with the femoral head core decompression group (P<.05). For unilateral lesions, postoperative Harris Hip Scores for all hips in the extracorporeal shock wave therapy group were more significantly improved than Harris Hip Scores for all hips in the femoral head core decompression group (P<.05). Patients who underwent extracorporeal shock wave therapy also resumed daily activities significantly earlier. Average overall operative time was similar in both groups. Symptoms disappeared significantly sooner in the extracorporeal shock wave therapy group in patients with both unilateral (P<.01) and bilateral lesions (P<.05). Hospital costs were significantly lower with extracorporeal shock wave therapy compared with femoral head core decompression. The intraoperative fluoroscopy radiation dose was lower in extracorporeal shock wave therapy than in femoral head core decompression for both unilateral (P<.05) and bilateral lesions (P<.01). On magnetic resonance imaging (MRI), bone marrow edema improved in all patients during the follow-up period. After extracorporeal shock wave therapy, all patients remained pain-free and had normal findings on posttreatment radiographs and MRI scans. Extracorporeal shock wave therapy appears to be a valid, reliable, and noninvasive tool for rapidly resolving intractable bone marrow edema syndrome of the hip, and it has a low complication rate and relatively low cost compared with other conservative and surgical treatment approaches.

Serum Biopterin and Neopterin Levels as Predictors of Empty Follicles.

OBJECTIVE: This study measured serum and follicular fluid (FF) levels of biopterin, neopterin, vascular endothelial growth factor (VEGF), and macrophage colony-stimulating factor (M-CSF) in patients receiving mild ovarian stimulation for oocyte retrieval. PATIENTS AND METHODS: Infertile patients who underwent ovarian stimulation were divided into the following: Group 1, no oocyte retrieval (n = 12), and Group 2, retrieval of more than four oocytes (n = 13). Median total gonadotropin dose in both groups was 150 IU. Biopterin and neopterin levels were measured using high-performance liquid chromatography. VEGF and M-CSF levels were measured by enzyme-linked immunosorbent assay. RESULTS: Compared to Group 2, serum and FF levels of neopterin and VEGF and serum levels of M-CSF were significantly increased, and serum and FF levels of biopterin were significantly decreased in Group 1 (P < 0.05 each). CONCLUSION: Biopterin and neopterin levels showed similar differences in FF and serum of patients with empty follicles. Decreased biopterin and increased neopterin in serum could predict poor oocyte retrieval.

Using a vortex mixer for testicular sperm collection.

OBJECTIVE: To evaluate 2 methods of processing testicular tissue for the retrieval of viable sperm from men with nonobstructive azoospermia. STUDY DESIGN: Fresh testicular tissue was obtained from nonobstructive azoospermia patients using a biopsy needle. The specimens were divided into 2 fractions. All specimens were minced and immersed in human tubal fluid (HTF). The first fraction was filtered through a nylon filter and incubated for 3 hours. The supernatant was centrifuged, resuspended in HTF and analyzed. The second fraction was immediately vortexed for 5 minutes and filtered through a nylon filter. The supernatant was centrifuged, resuspended in HTF and analyzed. RESULTS: Spermatozoa were obtained in 13 of 24 cases (54.2%) using the vortex method and in 5 of 24 cases (20.8%) with the nylon filter method. CONCLUSION: The vortex mixing method may be a better option than the conventional method for processing testicular tissue for sperm collection.

Expression and distribution of cyclooxygenase-2 in human ovary during follicular development.

Prostaglandins play important roles for oocyte maturation and follicular rupture. Cyclooxygenase-2 (COX-2), an inducible isoform of a prostaglandin metabolic enzyme, is essential for the timed production of prostaglandins in the ovary. The aim of the present study is to examine expression and distribution of COX-2 in human ovarian follicles at each of the maturation stages. Immunohistochemical staining shows that COX-2 is expressed in the granulosa cell layer of secondary and developing follicles, but is not detected in primary and Graafian follicles. Western blotting analysis revealed the existence of COX-2 in periovulatory follicular fluid at a mean concentration of 5.6 +/- 0.6 ng/ml. COX-2 might begin to be produced at the secondary follicle stage, and once a follicle reaches the Graafian follicle stage, the production of this enzyme is stopped. After exposure to luteinizing hormone (LH) surge, follicles might resume production of COX-2, and this is secreted into the follicular fluid.

Blackout during meals: A case report of swallow syncope due to sinus arrest.

A 79-year-old male, with a history of percutaneous coronary intervention (PCI), was referred to our cardiovascular department for a detailed examination of blackout caused by sinus arrest only during meals. Ultrasound echocardiography showed normal cardiac contraction with no asynergy, irrespective of the remaining stenotic coronary lesion. An electrophysiological study revealed deteriorated atrioventricular nodal conduction at a Wenckebach point of 70 beats per minute. However, sinus node function was normal as demonstrated by a sinus node recovery time of 1369 ms. Coronary angiography showed triple-vessel disease including the remaining stenotic coronary lesion, and a PCI was performed on the right coronary artery. Nevertheless, sinus arrest during meals was unchanged. Swallow syncope was partially improved by dietary modification; however, pacemaker implantation (PMI) was performed eventually, and the patient became asymptomatic after PMI. .

Three missense mutations of DNA topoisomerase I in highly camptothecin-resistant colon cancer cell sublines.

Various anticancer drugs, including camptothecins and indolocarbazoles, target DNA topoisomerase I (Top1). We previously described the camptothecin-resistant colon cancer cell line DLDSNR6, which has a Gly365Ser missense mutation in Top1. In the present study, we established highly camptothecin-resistant sublines from DLDSNR6 cells by continuous exposure to higher camptothecin concentrations. The established sublines grew in the presence of 30 µM of camptothecin, but exhibited markedly retarded growth. In addition to Gly365Ser, these sublines harbored a Top1 Gly717Arg mutation and some had also a Top1 Gln421Arg mutation. Top1 activity was reduced to approximately one-eighth in highly resistant cell lines compared with that in parental DLD-1 cells. Resistant clones with 3 Top1 mutations including Gln421RArg exhibited the highest resistance to the indolocarbazole J-107088 in terms of the effect on the cell cycle distribution. The Gln421 mutation was equivalent to a mutation recently found in camptothecin biosynthesizing plants, but it has not previously been found in mammalian cells.

In vitro effect of dehydroepiandrosterone sulfate on steroid receptors, aromatase, cyclooxygenase-2 expression, and steroid hormone production in preovulatory human granulosa cells.

OBJECTIVE: To examine the effect of a low concentration of DHEAS on the expression of the androgen receptor, estrogen receptor alpha and beta, progesterone receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2 in human preovulatory granulosa cells, and to measure their production of steroid hormones (estrone, estradiol, progesterone, androstenedione, and testosterone). DESIGN: Analysis of cultured primary human preovulatory granulosa cells by real-time reverse-transcriptase polymerase chain reaction and assays of hormone production. SETTING: Osaka City University Postgraduate School of Medicine. INTERVENTION(S): Preovulatory granulosa cells were collected from follicular fluid obtained from patients undergoing transvaginal oocyte retrieval with ultrasound guidance. The cells were cultured in the absence or presence of a low concentration of DHEAS. Real-time reverse-transcriptase polymerase chain reaction was performed to quantify the RNA expression of the investigated genes, and steroid hormone (estrone, estradiol, progesterone, androstenedione, and testosterone) levels were measured in the culture medium. MAIN OUTCOME MEASURE(S): Changes in [1] the levels of mRNAs encoding androgen receptor, estrogen receptor alpha, estrogen receptor beta, progesterone receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2; and [2] the levels of steroid hormones (estrone, estradiol, progesterone, androstenedione, and testosterone) in the culture medium. RESULT(S): Treatment of granulosa cells with 20 ng/mL DHEAS increased the expression of androgen receptor, aromatase, 3-beta-hydroxysteroid dehydrogenase, and cyclooxygenase-2, reduced the expression of estrogen receptor beta, and increased estrone and estradiol levels, but had no effect on progesterone, androstenedione, or testosterone levels. CONCLUSION(S): DHEAS may be an essential trigger of ovulation.

Insulin-lowering agents inhibit synthesis of testosterone in ovaries of DHEA-induced PCOS rats.

BACKGROUND: Insulin-lowering agents are reported to be useful in treating polycystic ovary syndrome (PCOS) anovulation. It has been suggested that lower insulin levels secondarily affect ovarian tissue, although the direct mechanism of action has not yet been verified. Here we investigated if these agents directly affect the ovary. METHODS: Thirty female Wister rats were studied. Six control rats were injected subcutaneously with 0.2 ml sesame oil, while 24 rats used as PCOS models were injected subcutaneously with dehydroepiandrosterone (DHEA) and divided into four groups. Six rats were injected with only DHEA, while the remaining 18 rats received metformin, pioglitazone or troglitazone. The ovaries were immunohistochemically stained with anti- testosterone and anti-17beta-HSD antibodies, and then evaluated for morphological changes. RESULTS: In the DHEA administration group, the number of atretic follicles significantly increased compared to that of control rats. The insulin-lowering agents did not improve the multicystic appearance. Serum testosterone concentrations significantly increased with DHEA administration, but the increase was inhibited by oral administration of insulin-lowering agents. Testosterone deposits in ovarian tissue were also reduced by feeding rats insulin-lowering agents. CONCLUSION: Insulin-lowering agents affected ovarian tissue by inhibiting testosterone biosynthesis in vivo.