Three-dimensional sensing of surfaces by projection of invisible electroluminescence from organic light-emitting diodes.

Organic light-emitting diodes (OLEDs) that efficiently emit near-infrared (NIR) light and consume little power will create valuable applications for OLEDs beyond just displays. Here, we report such a NIR-OLED with high operational stability that can be used as a light source for three-dimensional sensing of object's surfaces. Using a narrow-energy-gap material as a host for producing NIR hyperfluorescence system, we fabricated a NIR-OLED exhibiting intense emission at 930 nm with a high external electroluminescence quantum efficiency of more than 1% at a current density of 100 milliamperes per square meter without any degradation even after more than 300 hours of operation. The NIR-OLEDs were integrated with dense complementary metal-oxide semiconductor circuits to make a micro-NIR-OLED projector (0.21 inch, 230,400 pixels). By actively driving the projector on a pixel by pixel and projecting their emission onto objects, we successfully scanned and sensed the surfaces in three dimensions with invisible NIR.

A case of cystic lymphangioma arising from the parauterine tissue.

A 57-year-old woman, gravida 3, para 3, with no complaints visited our hospital for right-sided adnexal tumor found incidentally in cancer screening. She had no medical history, surgical history, or gynecological disease. Imaging studies showed a 5-cm lobular cystic tumor on the right side of uterus. We suspected right hydrosalpinx and decided to perform diagnostic laparoscopy. During laparoscopy, the right adnexa was found to be atrophic, and the tumor was located in the broad ligament. The tumor was observed to be a multilocular cyst containing yellow fluid that developed from the right parauterine tissue. The tumor was resected from the surrounding tissue. Histological examination revealed that the multilocular cyst contained a vascular component surrounding the lymphatic endothelium and was decided to be a cystic lymphangioma. The patient was followed up and there was no evidence of recurrence at postoperative 7 months. We experienced a very rare case of lymphangioma arising from the parauterine tissue. The laparoscopic approach can assist with both diagnosis and treatment.

NBS1 I171V variant underlies individual differences in chromosomal radiosensitivity within human populations.

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.

Regulation of REG4 Expression and Prediction of 5-Fluorouracil Sensitivity by CDX2 in Ovarian Mucinous Carcinoma.

BACKGROUND/AIM: The biological importance of the caudal-related homeobox transcription factor CDX2 in acquiring resistance to anticancer drugs has been studied in ovarian mucinous carcinoma. CDX2 promotes the expression of multidrug resistance 1 (MDR1) and confers resistance to paclitaxel. The regenerating islet-derived family member 4 (REG4) gene is a potential target gene of CDX2. In this study, we investigated the relationship between the expression of CDX2 and Reg IV and the regulation of Reg IV expression and examined novel chemotherapeutic regimens. MATERIALS AND METHODS: The regulation of Reg IV expression by CDX2 and sensitivity of 5-fluorouracil (5-FU) were evaluated using ovarian mucinous cancer cell lines. RESULTS: The correlation of CDX2 with Reg IV expression was demonstrated in ovarian mucinous carcinoma. Reg IV expression was enhanced by transfection of CDX2 and was suppressed by inhibition of CDX2 expression. OMC-3 cells with ectopically overexpressed CDX2 showed enhanced apoptosis and sensitivity to 5-FU. CONCLUSION: CDX2 promotes resistance to paclitaxel and sensitivity to 5-FU. Novel 5-FU-based chemotherapy based on CDX2 may be used in ovarian mucinous carcinoma.

Concurrent chemoradiotherapy for locally advanced squamous cell carcinoma of the cervix in a uterus didelphys with vaginal septum.

In November 2011, a 61-year-old woman was diagnosed with squamous cell carcinoma (SCC) of the cervix in a uterus didelphys with vaginal septum. The patient was diagnosed with Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage IIB because of infiltration to the left parametrium without infiltration to the pelvic wall. The patient was treated with external-beam radiotherapy (EBRT) and brachytherapy (BT), using concomitant chemotherapy with cisplatin. A total of 50 Gy were delivered (2 Gy/fraction/day) to the pelvis, with a central shield after 40 Gy. The patient was treated four times with BT (6 Gy × 4 fractions), with tandem and ovoid applicators inserted once to the left side; tandem to the left side and ovoid bilaterally were inserted twice; and tandem to the right side and ovoid bilaterally were inserted once. Six years and 8 months after the start of treatment, the patient had had no relapse or severe late adverse effects. For accurate diagnosis and optimal treatment of the uterus didelphys, careful interview and pelvic examination at initial diagnosis of a patient are very important.

Two Case Reports of Intravenous Leiomyomatosis with Hyaluronan Expression.

Intravenous leiomyomatosis (IVL) is a rare benign neoplasm. Herein, we describe two cases of IVL at different levels of progression. The tumor in Case 1 was extensive, invading the right atrium after a hysterectomy for a uterine myoma. The tumor temporarily responded to hormonal treatment; however, tumor regrowth occurred. In contrast, the tumor in Case 2 extended only to the pelvic veins and was revealed preoperatively. Hysterectomy and bilateral salpingo-oophorectomy were performed, resulting in the complete surgical resection of the tumor. In Case 2, no recurrence has been observed. Tumor samples were evaluated for hyaluronan expression using Alcian blue staining (with and without hyaluronidase digestion). The tumor in Case 1 stained strongly positive for hyaluronan while the tumor in Case 2 stained weakly positive for hyaluronan. In contrast, a large non-IVL uterine leiomyoma (control) stained negative for hyaluronan. These results suggest a relationship between tumor hyaluronan expression and IVL progression, similar to that in other cancers.

Regulation of multidrug resistance 1 expression by CDX2 in ovarian mucinous adenocarcinoma.

Epithelial ovarian cancer is an aggressive gynecological malignancy with a high mortality rate. Resistance against chemotherapeutic agents often develops in ovarian cancer patients, contributing to high recurrence rates. The multidrug resistance 1 (MDR1/ABCB1) gene encodes P-glycoprotein, which affects the pharmacokinetic properties of anticancer agents. We previously reported that the Caudal-related homeobox transcription factor CDX2 transcriptionally regulates MDR1 expression in colorectal cancer. CDX2 is a factor that influences cancer cell differentiation, malignancy, and cancer progression. We hypothesized that profiling of CDX2 and MDR1 expression could be an effective strategy for predicting anticancer drug resistance. We studied the expression of these factors in clinical samples from ovarian cancer patients. We found that endogenous MDR1 expression was positively associated with CDX2 expression in ovarian mucinous adenocarcinoma. Using ovarian mucinous adenocarcinoma cell lines, we also observed decreased MDR1 expression following inhibition of CDX2 by RNA interference. In addition, CDX2 overexpression in MN-1 cells, which display low endogenous CDX2, resulted in upregulation of MDR1 expression. CDX2 induced MDR1-dependent resistance to vincristine and paclitaxel, which was reversed by treatment with the MDR1-specific inhibitor verapamil. Our findings show that CDX2 promotes upregulation of MDR1 expression, leading to drug resistance in ovarian mucinous adenocarcinoma. Therefore, our study demonstrates the potential of novel chemotherapy regimens based on CDX2 status and MDR1 expression in ovarian mucinous adenocarcinoma.

Impact of UGT1A1 genotype upon toxicities of combination with low-dose irinotecan plus platinum.

AIM: Irinotecan-induced severe toxicities are possibly related to UGT1A1*6 and *28 genotypes. However, the correlation between UGT1A1 polymorphisms and the risk of toxicities induced by low-dose irinotecan plus platinum combination therapy still remains controversial. This prospective observational study aimed to examine the correlation between UGT1A1 genotypes and clinical outcomes of low-dose irinotecan (median 60 mg/m(2) , range 25-115 mg/m(2) ) plus platinum in Japanese patients with solid tumors. METHODS: Toxicity profiles were compared between UGT1A1 SNP heterozygotes (hetero-group) and patients with homozygous SNP profile (*6/*6, *28/*28 and *6/*28). Logistic regression models were used to identify independent risk factors for these toxicities. RESULTS: A total of 331 patients were enrolled: 84% with hetero-group and 16% with homo-group. Although the initial irinotecan dose was similar, the dose intensities during the three cycles were significantly lower in the homo-group (P < 0.01). Grade 3/4 hematological toxicities were significantly more frequent in the homo-group. Multivariable analysis identified UGT1A1 genotype (P < 0.01) as an independent factor for grade 4 hematological toxicity in the first treatment cycle. CONCLUSION: UGT1A1 genotype has a major impact on the increased risk of severe hematological toxicities, even in low-dose irinotecan regimens. UGT1A1 genotypes are useful biomarkers for predicting severe hematological toxicities in patients treated with irinotecan plus platinum analog.

Phase II study of concurrent chemoradiotherapy with weekly cisplatin and paclitaxel in patients with locally advanced uterine cervical cancer: The JACCRO GY-01 trial.

OBJECTIVE: A multicenter phase II trial was conducted to assess the efficacy and toxicity of concurrent chemoradiotherapy (CCRT) with weekly cisplatin (CDDP) and paclitaxel (PTX) in patients with locally advanced uterine cervical cancer. METHODS: Patients with FIGO stage III-IVA uterine cervical cancer without para-aortic lymphadenopathy were enrolled. Patients received definitive radiotherapy (RT) consisting of external beam whole-pelvic RT and high-dose-rate intracavitary brachytherapy. The cumulative linear quadratic equivalent dose was 62-65Gy prescribed at point A. weekly CDDP at 30mg/m(2) and PTX at 50mg/m(2) were administered concurrently with RT for ≥5 courses. RESULTS: Sixty-eight of the 70 registered patients were eligible. The complete response rate was 76.5% (95% confidence interval [CI], 66.4-86.6%). With a median follow-up of 27months (range, 7.9-33.5), the 2-year cumulative progression-free survival and the 2-year cumulative overall survival rates were 83.8% (95% CI, 75.1-92.6%) and 92.7% (95% CI, 86.4-98.9%), respectively. The pelvic cumulative disease progression-free and the 2-year cumulative distant metastasis rates were 89.6% (95% CI, 82.3-96.9%) and 13.2% (95% CI, 5.2-21.3%), respectively. The 2-year cumulative late complication rates were 25% for all grades, 13.2% for grade 1, 5.9% for grade 2, 2.9% for grade 3, and 2.9% for grade 4. CONCLUSIONS: For locally advanced cervical cancer, CCRT with weekly CDDP 30mg/m(2) and PTX at 50mg/m(2) demonstrated favorable antitumor activity, and was feasible and safe with respect to the protocol-specified serious adverse reactions and events. Evaluation of this regimen in phase III trials is warranted.

[Examination of taste threshold and serum zinc level change after chemotherapy].

For cancer patients undergoing chemotherapy, there is an onset of a variety of adverse events related to treatment. Among the adverse events at the moment is taste disorder, for which there is no established effective supportive care. We report the measurement and study their relationship across the changes in serum zinc and changes in the taste of patients undergoing chemotherapy. For cancer patients undergoing chemotherapy, taste threshold and serum zinc levels were measured on the day before administration of the therapeutic anti-cancer agent, and after administration of anticancer drugs on day 4 and day 7. Of taste thresholds in the test results, the threshold was salty on day 4 and day 7 after administration of anticancer agents, and a significant difference was found on day 7 after treatment with anticancer drugs on a day prior to administration of anticancer agents on day 1 (p<0. 001, p=0. 007), respectively. The serum zinc level was measured. There was no significant difference on day 7 after administration of anticancer agents and anti-cancer agent before administration on day 1 and day 7 after administration of anticancer drugs on day 4(p<0. 001, p<0. 05), respectively. A negative correlation was shown between the "salt of the fourth day threshold" and "serum zinc levels" (r=-0. 418, p

Intergender differences in histological architecture of the fascia pelvis parietalis: a cadaveric study.

The fascia pelvis parietalis (FPP) or endopelvic fascia is a well-known structure, but few studies described the detailed histological architecture, including the composite fiber directions. We hypothesized a gender-specific fiber architecture corresponding to the functional demand. For the first step to examine this hypothesis, we investigated specimens from 27 adult cadavers (10 males and 17 females) and 11 midterm fetuses (five males and six females) using immunohistochemistry and aldehyde-fuchsin staining. The adult female FPP was a solid, thick monolayered structure that was reinforced by abundant elastic fibers running across the striated muscle fibers, but it contained little or no smooth muscles (SM). In contrast, the male FPP was multilayered with abundant SM. In midterm fetuses, SM originated from the inferior part of the bladder and extended inferiorly along the gender-specific courses. Thus, we found a clear intergender difference in FPP architecture. However, the functional significance remained unknown because the basic architecture was common between nulliparous and multiparous women. Rather than for meeting the likely mechanical demands of pregnancy and vaginal delivery, the intergender difference of the FPP seemed to result from differences in the amount and migration course of bladder-derived SM as well as in hormonal background.

Comparative histological study of levels 1-3 supportive tissues using pelvic floor semiserial sections from elderly nulliparous and multiparous women.

AIM: The connective tissue located between the uterine cervix and sacrospinous ligament (the uterospinous connective tissue; USCT) has recently been noted as the level 1 supportive tissue instead of the classical uterosacral ligament. We examined whether or not the USCT changes its histological architecture by vaginal delivery in correlation with the levels 2 and 3 supportive tissues. METHODS: In the pelvic floors of 17 female cadavers (9 nuliparous and 8 multiparous), we compared histological architectures among the USCT, arcus tendineus fasciae pelvis (ATFP) and perineal membrane (PM). RESULTS: The USCT was evident as a string-like tissue structure in multiparous women or a thick mesh in nuliparous women. It consistently contained fewer elastic and smooth muscle fibers than other levels. In contrast, the ATFP usually contained abundant elastic fibers and smooth muscle. Likewise, the PM also displayed a constant morphology. CONCLUSION: Although all three sites were likely to be injured by delivery, the USCT seemed to be more severely damaged and/or more difficult to be recovered than the ATFP and PM.

Preclinical analysis of the antitumor efficacy of TS-1 using human uterine cervical cancer tumor xenografts.

We investigated the antitumor activity of TS-1 in comparison with that of UFT and cisplatin (CDDP) against cervical cancer using xenografts of a human uterine cervical squamous cell cancer cell line, CaSki, transplanted into female Balb/cA JcL-nu mice. CaSki cell xenografts were prepared by subcutaneous (s.c.) implantation of 3x10(6) cells/animal into the right dorsal region of the mice. The tumor volume was measured twice a week and the relative tumor volume (RTV) was calculated. We divided the animals into four groups according to the treatment administered; TS-1 (10 mg/kg orally, once daily for 14 consecutive days), UFT (24 mg/kg orally, once daily for 14 consecutive days), CDDP (7.6 mg/kg injected intravenously once on the 1st day) and control (no treatment) groups. The antitumor effects of the drugs were measured. On the 35th day after the completion of treatment, the mean tumor volume in the mice treated with TS-1 or CDDP changed from 132.873+/-11.783 mm(3) to 706.401+/-613.122 mm(3) and 133.809+/-19.366 mm(3) to 722.630+/-855.509 mm(3), respectively. The mean tumor volume in the groups treated with TS-1 or CDDP was significantly lower compared to that in the control group (p<0.001; one-tailed Student's t-test). The relative inhibition of the tumor growth was 65.31 in the TS-1 group, 48.31 in the UFT group and 64.51 in the CDDP group. We conclude that TS-1 administered orally for 14 consecutive days showed the highest antitumor activity.

UFT and its metabolite gamma-butyrolactone (GBL) inhibit angiogenesis induced by vascular endothelial growth factor in advanced cervical carcinoma.

OBJECTIVE: The aim of this study was to evaluate the potential role of UFT and its metabolite gamma-butyrolactone (GBL) for inhibition of angiogenesis induced by vascular endothelial growth factor (VEGF) in advanced cervical carcinoma by the determination of serum GBL and VEGF, and by immunohistochemical staining to assess VEGF protein expression, before and after UFT therapy. METHODS: The subjects were 35 patients with an advanced cervical carcinoma and five healthy volunteers between 2002 and 2003 at Hiroshima University Hospital, under informed consent. The patients received two courses of oral fluoropyrimidine (UFT) therapy at a dose of 600 mg/day for 5 and 2 days off treatment. Serum GBL and VEGF was measured before and after UFT therapy by the gas chromatography mass spectrometry and ELISA-kit in 22 patients and five healthy volunteers, respectively. Immunohistochemical detection of VEGF protein was done in 35 cervical cancers. Results The mean serum GBL level before and after UFT therapy was 21.9 +/- 2.3 and 79.3 +/- 6.2 ng/ml, respectively, and it was significantly increased after UFT administration (P < 0.0001). The mean serum VEGF level before and after UFT therapy was 95.3 +/- 28.1 and 67.5 +/- 11.2 pg/ml, respectively, and it was decreased by UFT administration. In 20 out of 33 (66.6%) patients who were detected with VEGF protein, VEGF protein expression was decreased by UFT therapy. The Delta GBL value (GBL after UFT--GBL before UFT therapy) showed a significant inverse correlation with Delta VEGF value (VEGF after therapy--VEGF before therapy) (r2 = 0.940). CONCLUSIONS: Our findings suggest that UFT and its metabolite GBL inhibit angiogenesis induced by VEGF to have an antitumor effect on cervical cancer.

[Eco-hydrological characteristics of a subtropical evergreen broadleaved forest in Japan].

A three years (1998-2000) observation on the eco-hydrological characteristics of Castanopsis sieboldii and Schima wallichii dominated subtropical evergreen broadleaved forest on the Okinawa Island of Japan showed that during the observation period, the mean annual precipitation was 3325 mm, and 42.3% of it was directly or indirectly contributed by typhoon rainfall, implying that typhoon played a very important role in the hydrology of the forest. Through-fall and stem-flow contributed to 53.9% and 30.9% of the annual rainfall, respectively. The higher stem-flow could be due to the high wind velocity, intense rainfall, and the crown morphology of dominant species C. sieboldii with inclined branches and concave-shaped leaves. Interception loss occupied 15.2% of the total rainfall, being the lowest in the range of 15%-30% widely reported for many broadleaved evergreen forests. The annual surface runoff and lateral flow were 1092 mm and 613 mm, respectively, and the high proportion of surface runoff (32.8%) was probably due to the poor physical properties of soil, particularly the low noncapillary porosity (8%-12%), high clay content (51.9%-60.5%), and low infiltration rate (12 ml x min(-1)) in deeper soil layers. The effective and maximum water storage capacity of 0-70 cm soil layer were 85 mm and 324 mm, respectively, and the maximum water-holding capacity of forest floor was 2. 8 mm, which were close to or lower than those of the subtropical and tropical rain forests in China.

Gynecologic abscess: CT-guided percutaneous drainage.

A 42-year-old woman with recurrent bilateral endometrial ovarian cystoma presented with fever and pelvic pain caused by a tubo-ovarian abscess (TOA), which was resistant to several varieties of intravenous and oral antibiotics for 2 weeks (Case 1). Computed tomography (CT)-guided diagnostic aspiration for a rapid enlarged right ovarian cystoma through a transabdominal route confirmed that it had developed into a TOA. Subsequent percutaneous abscess drainage (PAD) and irrigation for 3 days were successful. One-year follow-up revealed no recurrence of TOA. A 58-year-old woman with recurrent cervical cancer after external radiation therapy (RT) presented with fever, confusion and tremor caused by pyometra (Case 2). Since transvaginal drainage was impossible due to cervical os obstruction, the patient had undergone CT-guided transabdominal PAD and irrigation for a month. Thereafter, the clinical findings improved and a tracheloplasty was performed to prevent recurrence. CT-guided PAD may be a useful treatment option for gynecologic abscess as a diagnostic aspiration, a temporizing procedure until surgery, or an alternative surgery.

5-O-glucosyldihydroflavones from the leaves of Helicia cochinchinensis.

From the leaves of Helicia cochinchinensis, collected on Okinawa Island, seven phenolic glucosides and two terpenic glucosides were isolated. Five of the phenolic glucosides were previously known, being identified with p-coumaric and ferulic acids glucosyl esters, rhodioloside, helicidiol, and naringenin 5-O-beta-D-glucopyranoside. The structures of two other phenolic glucosides, named heliciosides A and B, were elucidated to be 5-O-beta-D-glucosides of 3-hydroxyflavanone, namely aromadendrin and taxifolin, by means of spectroscopic analyses. The two terpenic glucosides were identified with ampelopsisionoside and icariside C1.

Predicting the chemosensitivity of ovarian and uterine cancers with the collagen gel droplet culture drug-sensitivity test.

We investigated the utility of the collagen gel droplet culture drug-sensitivity test (CD-DST) for predicting the response of gynecological cancers to chemotherapy. Eighty-three cancer patients were enrolled in this study: 26 ovarian, 29 cervical and 31 endometrial cancers. The CD-DST was performed at various concentrations of drugs. We calculated the T/C ratio, where T is the total volume of the treated culture and C is the total volume of the control culture, and a T/C ratio of 50% or less was defined as sensitive in vitro. The efficacy rate (%) was defined as the number of cultures with a T/C ratio of 50% or less, divided by the total number of evaluable cultures. True-positive cases were defined as clinical responders (complete+partial responses) and true-negative cases were defined as clinical non-responders. The overall tumor evaluation rate was found to be 79.1%. The appropriate drug concentrations were selected as 1.0 microg/ml for cisplatin, 20.0 microg/ml for carboplatin, 1.0 microg/ml for paclitaxel and 0.1 microg/ml for docetaxel by the linear regression equations. The in vitro sensitivity for each drug showed a significant correlation with clinical response rates (r=0.592, p=0021). We therefore conclude that the CD-DST can be used to predict the response to anti-cancer drugs, and may also provide important information by contributing to the development of new chemotherapy regimens.

Lanceocrepidiasides A-F, glucosides of guaiane-type sesquiterpene from Crepidiastrum lanceolatum.

From the aerial parts of Crepidiastrum lanceolatum, six guaiane-type sesquiterpene glucosides, lanceocripidiasides A-F were isolated together with five known sesquiterpene glucosides, ixerin Y, crepidialanceosides A and B, and youngiasides A and D, two known megastigmane glucosides, icariside B1 and corchoionoside A, and benzyl 6'-O-beta-D-apiofuranosyl-beta-D-glucopyranoside. Structures were elucidated by spectroscopic analyses.

Expression of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in uterine endocervical glands and in cervical adenocarcinomas.

To investigate the potential role of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in the development and progression of uterine cervical adenocarcinoma, we examined hK7 and ALP protein expression by immunohistochemistry in 70 cervical adenocarcinomas and 13 normal cervical tissues. Positive hK7 expression rates in normal endocervical glands and in cervical adenocarcinomas were 46.2 and 80%, respectively. A significantly higher hK7 expression rate was observed in cervical adenocarcinomas compared to normal endocervical glands (p=0.0099). In contrast, positive ALP detection rates in normal endocervical glands and in cervical adenocarcinomas were 100 and 15.7%, respectively. A significantly lower ALP detection rate was observed in cervical adenocarcinomas compared to normal endocervical glands (p<0.0001). There was a significant inverse correlation between hK7 and ALP expression status (p=0.0010). However, no statistically significant differences in hK7 or ALP expression status were found with respect to age, clinical stage, histological grade and lymph node metastasis status in cervical adenocarcinoma cases. Log-rank testing showed that advanced clinical stage and positive lymph node metastasis significantly correlated with poor patient survival (p=0.0005 and p<0.0001, respectively), whereas no correlation was found between hK7 or ALP expression and survival. These results suggest that increased expression of hK7 and decreased expression of ALP might play an important role in cervical adenocarcinoma development.