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Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.

5529 Background: LACvCa has a poor prognosis. CCRT is the standard treatment for LACvCa, and the 5-year survival rate is estimated at around 60%. Nivolumab (Nivo), an anti-PD1 monoclonal antibody, showed clinical activity in recurrent or persistent CvCa pts. Nivo may enhance antitumor immune responses induced by CCRT. The safety and feasibility of Nivo plus CCRT for LACvCa pts has not yet been reported. We report data from a phase I trial evaluating safety and feasibility of pre- and co-administration of Nivo with CCRT in pts with LACvCa (GOTIC-018; JMA-IIA00425). Methods: The treatment plan in cohort A is co-administration of Nivo (240mg/body once every 2 weeks) with CCRT followed by maintenance therapy with Nivo. The treatment plan in cohort B is pre- (two doses of Nivo before CCRT) and then co-administration of Nivo with CCRT followed by Nivo maintenance. The CCRT regimen includes 4 or more cycles of cisplatin (40 mg/m2 weekly) and external beam radiotherapy (EBRT) followed by brachytherapy. The Nivo maintenance therapy was scheduled for 52 weeks after completion of CCRT. The primary objective is the rate of Grade≧3 adverse events (AEs) during the acute phase, which is defined as 90 days from the start date of EBRT. Secondary objectives include the incidence of dose limiting toxicity (DLT) and progression-free survival. Results: A total of 30 patients, 15 patients in each cohort, was enrolled in this study. This report is a safety evaluation in the acute phase of the study. There were 1 stage IVA, 11 stage IIIB, 16 stage II and 2 stage IB tumors based on FIGO 2009. 28 squamous cell and 2 adeno/adenosquamous carcinomas were included. No DLT was observed in the first 6 DLT-evaluable pts in each cohort. All 30 patients completed planned EBRT and brachytherapy. 2 and 0 patients required a break from EBRT in cohort A and B, respectively. The median cycles of cisplatin administration was 5 and 6 in cohort A and B, respectively. 2 and 0 patient required cisplatin dose reduction in cohort A and B, respectively. 1 patient required cisplatin discontinuation in each cohort. The median cycles of Nivo administration were 6 and 9 in cohort A and B, respectively. 1 patient required Nivo discontinuation due to AEs in each cohort. All patients experienced Grade≧3 AEs. Most common Grade≧3 AEs were neutropenia (60.0 and 26.7% in cohort A and B, respectively), anemia (13.3 and 16.7%) and diarrhea (13.3 and 26.7%). In cohort B, no patients required delay in starting CCRT due to the AEs related to pre-administration of Nivo, and no patients had disease progression before starting CCRT. Conclusions: No DLT was reported during the acute phase in both cohort A and B, and no new safety signals were observed. Addition of pre-and co- administration of Nivo appears safe and feasible in patients with LACvCa treated with CCRT. Clinical trial information: JMA-IIA00425.

BackgroundRecent phase III randomized trials have suggested that neoadjuvant chemotherapy followed by interval debulking surgery (NACT-IDS) is a treatment option for patients with advanced epithelial ovarian cancer. This study aimed to use CA-125 and computed tomography (CT) scanning to generate a simple and clinically applicable model of predicting complete cytoreduction by interval debulking surgery (IDS) and the overall survival in patients who receive taxane/platinum-based chemotherapy as neoadjuvant chemotherapy (NACT).MethodsPatients with stage IIIc or IV epithelial ovarian cancer who underwent taxane/platinum-based NACT followed by IDS in Gunma Prefectural Cancer Center, Takasaki General Medical Center, and Gunma University from April 2009 to March 2015 were included. Patients underwent a CT scan to confirm confirm tumors unresectable by standard surgery before NACT. CA-125 levels were measured pre-NACT, after each cycle of NACT, and before IDS. CT was also performed before IDS to evaluate tumor metastasis. Data were collected retrospectively and analyzed to determine the predictive factors of complete resection and overall survival.ResultsAmong 63 patients who received NACT-IDS, 43 and 20 patients had stages IIIc and IV epithelial ovarian cancer at diagnosis, respectively. CT predictors of residual tumors after IDS such as extra-ovarian implants (P= 0.009) and omental cakes (P= 0.038) were not present. Univariate analysis revealed that the independent factors for overall survival were no residual tumor by IDS (P= 0.0016) and CA125 ≤ 20 U/ml before IDS (P= 0.0011).ConclusionsAlthough this study had a small sample size, NACT-IDS used to completely remove macroscopic disease which significantly improved the prognosis of patients with preoperative CA-125 ≤ 20 U/ml. Results from this study provide useful information for future studies on the management of patients with advanced epithelial ovarian cancer.

ObjectiveThe ubiquitin C-terminal hydrolase L1 (UCHL1) plays a key role in tumor invasion and metastasis. Ubiquitin C-terminal hydrolase L1 is overexpressed in various cancers and reported to be correlated with a poor prognosis. The objective of this study was to determine the prognostic significance of UCHL1 in endometrial cancer.MethodsThe expression of UCHL1 in endometrial cancer was assessed using quantitative reverse transcription polymerase chain reaction and immunohistochemistry in 56 and 215 resected tumor specimens, respectively.ResultsThe 4-year survival rates of the high UCHL1 messenger RNA expression group and high UCHL1 protein expression group were 78% and 71%, respectively, compared with 96% and 95% for the low UCHL1 messenger RNA expression group and low UCHL1 protein expression group, respectively. Kaplan-Meier and log-rank tests indicated a significant correlation between expression of UCHL1 and disease-free survival and overall survival. Moreover, multivariate stepwise Cox proportional hazard regression model analysis showed that UCHL1 was a significant independent marker for predicting a poor disease-free survival and overall survival. In 43 patients with metastatic lesions, immunohistochemical analysis of metastatic lesions revealed that the recurrence rate and mortality rate were 62% and 41%, respectively, in 29 UCHL1-positive patients and 36% and 29%, respectively, in 14 UCHL1-negative patients.ConclusionsThe results of this study suggest that high UCHL1 expression is a strong marker of poor prognosis of endometrial cancer. Furthermore, we suggest that UCHL1 may be involved in the development of distant metastasis in endometrial cancer.

Fan-out package technology for realizing high density and high integration is promising for 3D integration. This paper presents an innovative 3D fan-out packaging technology with photosensitive through mold interconnects. This is the first-time demonstration of 3D integration based on fan-out technology using a photosensitive mold compound material to provide high-aspect-ratio and fine-pitch through mold via openings, thereby advanced substrate-free packaging. 10 × 10 mm Si test dies with 100-µm thickness was prepared to confirm feasibility of the 3D integration packaging process. The chip design comprises over 2000 pads with a 200-µm pad pitch. After embedding the test die into a thick photosensitive molding compound, the lithography process conditions for shallow vias on the die and deep visa around the die were evaluated to realize a 3D integration packaging without conventional tall electroplated Cu pillars. The prototype package with photosensitive through mold interconnects shows a decent reliability over a 1000-cycle package-level thermal cycle test, a 1000-h high-temperature storage test, and a 96-h pressure cooker test. Thermal stress simulation has revealed that package warpage with the photosensitive material is small compared with that of a widely used epoxy molding compound for chip first fan-out wafer-level package. This study demonstrates future viability of the fan-out process using a photosensitive molding compound to enhance the versatility of 3D integration, thereby dramatically expanding the application field for fan-out packages.

This paper deals with the process integration to produce a high performance processor with a large-scale and high-bandwidth DRAM chip stacked. Ten micrometres pitch Cu redistribution wiring has been implemented on 12 in. DRAM wafers to relocate the Al bond pads where [email protected] pitch SnCu bumps are formed for a large number of I/Os of the memory interface. A phenol-melamine based resin film which is curable at less than 250^oC can be used so as to avert detraction from memory retention yield as well as to insulate the Cu lines, leading to reduction of wafer warpage in an effort to enable fine pitch lithography. In a chip-on-chip joining process, the logic chip is interconnected with the DRAM chip with a less than [email protected] accuracy by mass reflow bonding of the SnCu micro-bumps on both of the chips, revealing the self-aligning effect of the solder bumps. No failures have been observed after reliability stressing on the packaged two-chip stack. The present process integration has been qualified for the mass producing line to provide some merchant LSI devices and will be viable to future generation devices with more chips stacked using through Si via technology.

Background The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, has been clinically applied for the treatment of a variety of tumors with EGFR overexpression. A phase II clinical study of erlotinib (NCIC IND-148) for recurrent or metastatic endometrial carcinoma (EC) resulted in an unfavorable result. However, in that study, the expression levels of EGFR were not accurately analyzed. Thus, the aim of this study was to re-examine the efficacy of erlotinib in EC cells by utilizing in vitro and in vivo models. Methods Tissue samples obtained from patients histologically diagnosed with EC of the uterine corpus were subjected to immunohistochemistry and RT-PCR to determine the protein and mRNA expression levels of EGFR. Western blot and WST-1 assays of EGFR siRNA-transfected HEC-1A, KLE, and Ishikawa cells were used to evaluate the efficacy of erlotinib in tumor cell lines expressing different EGFR levels. Furthermore, HEC-1A and Ishikawa cells were implanted into athymic mice treated with either erlotinib or trastuzumab. Results At our institution, 20.9 % of endometrial cancer patients with low grade endometrioid histology have been diagnosed as stage III and IV. Immunohistochemical analysis and RT-PCR revealed the presence of significant EGFR and EGFR mRNA expression in low-grade endometrioid carcinoma in comparison with high-grade endometrioid carcinoma. In vitro study, WST-1 assay and Western blot analysis revealed that EGFR expression levels were correlated with tumor cell viability. Erlotinib reduced the proliferation of HEC-1A expressing high levels of EGFR, while trastuzumab showed similar effect in Ishikawa cells dominantly expressing human epidermal growth factor receptor type2 (HER2). In vivo erlotinib decreased tumor growth in mice xenografted with HEC-1A cells, whereas this tumor-growth inhibition was not observed in trastuzumab-treated mice xenografted with Ishikawa cell. Conclusions EGF contributed to tumor proliferation in EC cell lines along with EGFR expression in vitro. Erlotinib also demonstrated anti-tumor effects in xenograft mice models. Our results suggest that erlotinib continues to have clinical usefulness in specific cases, after taking into consideration the EGFR expression levels.

Estrogen has been considered to enhance FSH actions in the ovary, including the induction of the LH receptor (LHR). In this study, we elucidated the mechanism underlying the effect of estrogen on the induction of LHR by FSH in rat granulosa cells. Estradiol clearly enhanced the FSH-induced LHR mRNA increase in a time- and dose-dependent manner, with a maximum increase of approximately 3.5-fold at 72 h, compared with the level of LHR mRNA solely induced by FSH. We then investigated whether the effect of estrogen on LHR mRNA was due to increased transcription and/or altered mRNA stability. A luciferase assay with the plasmid containing the LHR 5′-flanking region did not show that estradiol increased the promoter activity induced by FSH. In contrast, the decay curves for LHR mRNA showed a significant increase in half-life with FSH and estradiol, suggesting that the increased stability of LHR mRNA is at least responsible for the regulation of LHR mRNA by estrogen. Recently mevalonate kinase (Mvk) was identified as a trans-factor that binds to LHR mRNA and alters LHR mRNA stability in the ovary. We found that estradiol, with FSH, decreased Mvk mRNA levels in rat granulosa cell culture, resulting in up-regulation of LHR mRNA that was inversely correlated to Mvk mRNA expression. Furthermore, the augmentation of FSH-induced LHR expression in the presence of estrogen was erased with the overexpression of Mvk by transient transfection. Taken together, these data indicate that LHR mRNA is up-regulated due to increased stability when estrogen negatively controls Mvk.

Lymphangioleiomyomatosis (LAM) is a rare, systemic disorder that predominantly affects women. Although patients with LAM mostly present with pulmonary symptoms, some patients may present initially with extrapulmonary symptoms. We present a case of a 30-year-old Japanese female with abdominal pain during menstrual periods was suspected of having ovarian cancer due to exaggerated ascites observed at a local clinic. Subsequently, she was transferred to our hospital for further investigations, and was diagnosed with LAM. Three years after diagnosis, she had a girl by cesarean section to avoid the progression of pulmonary LAM by vaginal delivery. The patient is undergoing follow-up treatment with the administration of gonadotropin-releasing hormone-analog. Though LAM is rare, gynecologists should know about it because it may occur with gynecological symptoms in young women.

MAPK kinase (MEK)1 and MEK2 were deleted from Leydig cells by crossing Mek1(f/f);Mek2(-/-) and Cyp17iCre mice. Primary cultures of Leydig cell from mice of the appropriate genotype (Mek1(f/f);Mek2(-/-);iCre(+)) show decreased, but still detectable, MEK1 expression and decreased or absent ERK1/2 phosphorylation when stimulated with epidermal growth factor, Kit ligand, cAMP, or human choriogonadotropin (hCG). The body or testicular weights of Mek1(f/f);Mek2(-/-);iCre(+) mice are not significantly affected, but the testis have fewer Leydig cells. The Leydig cell hypoplasia is paralleled by decreased testicular expression of several Leydig cell markers, such as the lutropin receptor, steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 17α-hydroxylase, and estrogen sulfotransferase. The expression of Sertoli or germ cell markers, as well as the shape, size, and cellular composition of the seminiferous tubules, are not affected. cAMP accumulation in response to hCG stimulation in primary cultures of Leydig cells from Mek1(f/f);Mek2(-/-);iCre(+) mice is normal, but basal testosterone and testosterone syntheses provoked by addition of hCG or a cAMP analog, or by addition of substrates such as 22-hydroxycholesterol or pregnenolone, are barely detectable. The Mek1(f/f);Mek2(-/-);iCre(+) males show decreased intratesticular testosterone and display several signs of hypoandrogenemia, such as elevated serum LH, decreased expression of two renal androgen-responsive genes, and decreased seminal vesicle weight. Also, in spite of normal sperm number and motility, the Mek1(f/f);Mek2(-/-);iCre(+) mice show reduced fertility. These studies show that deletion of MEK1/2 in Leydig cells results in Leydig cell hypoplasia, hypoandrogenemia, and reduced fertility.

Lymphangioleiomyomatosis (LAM) is a rare, systemic disorder that predominantly affects women. Although patients with LAM mostly present with pulmonary symptoms, some patients may present initially with extrapulmonary symptoms. We present a case of a 30-year-old Japanese female with abdominal pain during menstrual periods was suspected of having ovarian cancer due to exaggerated ascites observed at a local clinic. Subsequently, she was transferred to our hospital for further investigations, and was diagnosed with LAM. Three years after diagnosis, she had a girl by cesarean section to avoid the progression of pulmonary LAM by vaginal delivery. The patient is undergoing follow-up treatment with the administration of gonadotropin-releasing hormone-analog. Though LAM is rare, gynecologists should know about it because it may occur with gynecological symptoms in young women.

Leading-edge LSI products with 40nm logic technology node and beyond are facing the issue of how higher memory bandwidth is reconciled with lower power consumption. Chip stacking of a logic chip on a large-scale DRAM chip, interconnected with each other by fine-pitch bumps, provides a solution to realize a power efficient SiP (System in Package). In this paper, the successful process integration of 10µm pitch Cu redistribution wiring and 40µm pitch SnCu micro-bumping on 300mm wafers, together with chip-on-chip (CoC) joining, has been described in an effort to relinquish embedded DRAM (eDRAM) SoC (System on Chip).

This paper elucidated for the first time that titanium (Ti) is an excellent barrier metal (BM) material from the stand point of cost and performance, especially for the porous low-k ILD materials. Both stress induced voiding (SIV) suppression and one order longer electromigration (EM) lifetime were obtained by introducing Ti instead of the conventional tantalum (Ta). It has been considered that the smaller volume change when oxidized and the existence of metallic Ti-O solid-solution phase for Ti would be the reason for its control of moisture penetration from the low-k ILD materials which resulted in excellent SIV suppression. No electrical resistance increase due to intermetallic reaction between Cu and Ti was observed. Furthermore, the suppression of Cu grain boundary migration was attributed to the segregation of Ti atoms at the Cu grain boundaries. This resulted in higher interconnect reliability

A splice variant of human lutropin/choriogonadotropin-receptor [hLHR (exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a regular menstrual-cycle. Supported by detergent soluble binding assay and receptor biotinylation experiment, the receptor binding assay shows hLHR (exon 9) is neither expressed at the cell surface nor have the capability of binding to hCG. Interactions between hLHR (exon 9) with the immature bands of gonadotropin receptors not with the mature bands were seen. This phenomenon is specific among gonadotropin receptors since human thyrotropin-receptor (hTSHR) failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the receptor protein level within the cells. To elucidate the mechanism underlying the decrease in receptor protein by this receptor complex, we performed a Percoll-fractionation experiment, which indicated the receptor complex drove hLHR to the lysosome instead of the plasma-membrane. Moreover, the expression of hLHR (exon 9) mRNA was seen at all phases of the menstrual cycle and relatively increased as the luteal phase progressed. These results reveal a novel mechanism of regulation for gonadotropin receptor expression.

A splice variant of human lutropin (LH)/choriogonadotropin (CG)-receptor [hLHR(exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a normal menstrual cycle. Supported by a detergent-soluble binding assay and a receptor biotinylation experiment, the receptor binding assay shows hLHR(exon 9) is neither expressed at the cell surface nor has the capability of binding to hCG. In addition, hLHR(exon 9) was confirmed in the endoplasmic reticulum (ER) by endoglycosidase H treatment. A coimmunoprecipitation experiment clearly showed that hLHR(exon 9) and constitutively inactivate mutant-LHRs, which stay in the ER, form an association with the human follitropin (FSH)-receptor (hFSHR). This suggests that in the presence of mutant-LHR, hFSHR, which is trapped in the ER and associated with hLHR(exon 9), is unable to come up to the plasma membrane. This phenomenon is specific among gonadotropin receptors because human TSH receptor failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the hFSHR receptor protein level within the cells, which impaired cAMP production. To elucidate the mechanism underlying the decrease in hFSHR protein by this receptor complex, we performed a Percoll fractionation experiment, which indicated that the receptor complex drove hFSHR to the lysosome instead of the plasma membrane. These results reveal a novel mechanism of FSHR expression regulation.

We previously reported a splice variant form of human LH receptor [hLHR(exon 9)] that lacks exon 9, coding the N-terminal extracellular region close to the first transmembrane domain. Several recent studies suggest that G protein-coupled receptors are able to form dimerization or oligomerization of the receptor, suggesting an intermolecular interaction between hLHR(exon 9) and the wild-type LH receptor (hLHR). The aim of this study, using coimmunoprecipitation, is to examine whether hLHR forms an association with hLHR(exon 9). An interaction between hLHR(exon 9) with the immature band (68 kDa) of hLHR and not with the mature band (85 kDa) was seen. When hLHR and hLHR(exon 9) were coexpressed, the density of hLHR expression was significantly reduced, compared with hLHR expressed alone. The human chorionic gonadotropin-stimulated cAMP accumulation in the cells expressing hLHR(exon 9) was also impaired, compared with the cells expressing hLHR. In this study, we demonstrated that hLHR is capable of forming receptor complexes. Our findings may expand the possibility of a splice variant of hLHR specifically modulating the functional property of the wild-type hLHR.