The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae.

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5'-CGGCTAATGCGG-3', and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

Bioinformatics in bioscience and bioengineering: Recent advances, applications, and perspectives.

Recent advances have led to the emergence of highly comprehensive and analytical approaches, such as omics analysis and high-resolution, time-resolved bioimaging analysis. These technologies have made it possible to obtain vast data from a single measurement. Subsequently, large datasets have pioneered the data-driven approach, an alternative to the traditional hypothesis-testing system, for researchers. However, processing, interpreting, and elucidating enormous datasets is no longer possible without computation. Bioinformatics is a field that has developed over long periods, intending to understand biological phenomena using methods collected from information science and statistics, thus solving this proposed research challenge. This review presents the latest methodologies and applications in sequencing, imaging, and mass spectrometry that were developed using bioinformatics. We presented the features of individual techniques and outlines in each part, avoiding the use of complex algorithms and formulas to allow beginning researchers to understand an overview. In the section on sequencing, we focused on comparative genomic, transcriptomic, and bacterial microbiome analyses, which are frequently used as applications of next-generation sequencing. Bioinformatic methods for handling sequence data and case studies were described. In the section on imaging, we introduced the analytical methods and microscopy imaging informatics techniques used in animal cell biology and plant physiology. We introduce informatics technologies for maximizing the value of measured data, including predicting the structure of unknown molecules and untargeted analysis in the section on mass spectrometry. Finally, we discuss the future outlook of this field. We anticipate that this review will assist biologists in using bioinformatics more effectively.

Comprehensive investigation of the gene expression system regulated by an Aspergillus oryzae transcription factor XlnR using integrated mining of gSELEX-Seq and microarray data.

BACKGROUND: Transcription factors (TFs) specifically bind to DNA sequences and control the expression of target genes. AoXlnR is a key TF involved in the expression of xylanolytic and cellulolytic enzymes in the filamentous fungi, Aspergillus oryzae. Genomic SELEX-Seq (gSELEX-Seq) can reveal the in vitro binding sites of a TF in a genome. To date, the gene expression network controlled by AoXlnR in A. oryzae is not fully explored. In this study, the data from gSELEX-Seq analysis and data mining were applied toward a comprehensive investigation of the AoXlnR-regulated transcriptional network in A. oryzae. RESULTS: Around 2000 promoters were selected as AoXlnR-binding DNAs using gSELEX-Seq, consequently identifying the genes downstream of them. On the other hand, 72 differentially expressed genes (DEGs) related to AoXlnR had been determined by microarray analysis. The intersecting set of genes, that were found using the gSELEX-Seq and the microarray analysis, had 51 genes. Further, the canonical AoXlnR-binding motifs, 5'-GGCT(A/G) A-3', were successfully identified in gSELEX-Seq. The motif numbers in each promoter of the DEGs and differential expression levels were correlated by in silico analysis. The analysis showed that the presence of both 5'-GGCTAA-3' and 5'-GGCTGA-3' motif has significantly high correlation with the differential expression levels of the genes. CONCLUSIONS: Genes regulated directly by AoXlnR were identified by integrated mining of data obtained from gSELEX-Seq and microarray. The data mining of the promoters of differentially expressed genes revealed the close relation between the presence of the AoXlnR-binding motifs and the expression levels of the downstream genes. The knowledge obtained in this study can contribute greatly to the elucidation of AoXlnR-mediated cellulose and xylan metabolic network in A. oryzae. The pipeline, which is based on integrated mining of data consisting of both in vitro characterization of the DNA-binding sites and TF phenotype, can be a robust platform for comprehensive analysis of the gene expression network via the TFs.

Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay.

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions. Furthermore, these DNA-binding tags were used to establish an enzyme assay system based on the spatial arrangement of transglutaminase and its substrate at the molecular level. Together, the results of the present study suggest that the scCro-tag may be a powerful tool to facilitate the synthetic spatial arrangement of proteins on a DNA ligand.

[Laparoscopic partial nephrectomy for renal leiomyoma: a case report].

A 60-year-old man visited our hospital with a complaint of right renal incidentaloma which was pointed out on abdominal ultrasonography for a medical check-up. Abdominal computed tomography showed a renal tumor in the right kidney, which was a slightly high-dense relative to the renal parenchyma and was enhanced in the arterial phase. The tumor had grown gradually from 1.4 to 1.7 cm in diameter. After the observation for 4 years, he underwent pure laparoscopic non-ischemic partial nephrectomy using a microwave tissue coagulator. Histological examination of the specimen revealed a leiomyoma of the kidney. This is the 5th case of successful laparoscopic partial nephrectomy for renal leiomyoma in Japan within the retrieved references.

[A case of small cell carcinoma of the renal pelvis].

A 77-year-old man visited our hospital with a chief complaint of asymptomatic gross hematuria. He was diagnosed with right renal pelvic tumor (7 cm) involving right renal hilar and inter-aortocaval lymph node metastases by radiological evaluation, and cytologic examination of urine indicated small cell carcinoma. After neoadjuvant chemotherapy with gemcitabine and cisplatin, right nephroureterctomy with bladder cuff, and right renal hilar and inter-aortocaval lymph node dissection was performed. Histological examination of the specimen revealed a small cell carcinoma of the renal pelvis (ypT3N2). After the operation, adjuvant chemotherapy with etopside and carboplatin was administered in combination with radiation therapy. At 5 months after the operation, there has been no evidence of recurrence. To our knowledge, this is the 38th report of a small cell carcinoma originating from the kidney in the literature.

[Multi-institute survey on actual conditions of urologic management for severe bladder dysfunction after hysterectomy].

We conducted a multi-institute survey on the conditions related to urologic management of severe voiding dysfunction after hysterectomy for uterine cancer with or without postoperative irradiation. Our first study population was a group of adult female patients currently managed by urologists, using clean intermittent catheterization (CIC). Of the 287 patients in this group, 99 (34%) had suffered from uterine cancer. Of these patients, 94 underwent hysterectomy for this disease; 44 and 30 were treated with or without postoperative radiation, respectively, while postoperative irradiation status was unknown for 20. Median follow-up after surgery was 21 (0.2-52) years and median interval from operation to the introduction of CIC was 4.0 (0-49) years. CIC tended to be introduced later for patients with postoperative radiation than those without it. Seventy-four patients, who required invasive urologic interventions other than CIC for voiding dysfunction after hysterectomy, are the second study population. Most of these (82%) had received postoperative irradiation. Continuous Foley catheter placement was the most frequent procedure. Long-term follow-up and urologic management for voiding dysfunction is required for patients undergoing hysterectomy.

Two cases of chyloretroperitoneum following retroperitoneoscopic nephroureterectomy.

Post-operative chyloretroperitoneum is a rare complication in urological surgery. We experienced two cases of post-operative chyloretroperitoneum following retroperitoneoscopic left nephroureterectomy. Both cases were treated successfully with drainage, low fat diet, total parenteral nutrition (case 1 only), sclerotherapy and somatostatin analog. Laparoscopic procedures are supposed to have a higher risk of chylous complications, as CO2 pressure appears to make the operator overlook suppressing the lymphatic leakage from the resected lymphatic channels.

[Clinical review of conservative management of upper urinary tract transitional cell carcinoma].

We reviewed 18 patients with transitional cell carcinoma of the renal pelvis and ureter undergoing nephron-sparing surgery between April 1990 and Febrary 2003. The mean age of the patients, 17 males and one female, was 69 years (range 33-88 years). The tumor site was the renal pelvis in 2, ureter in 13 and ureteral orfice in 2. Six of them were imperative cases and 12 were elective. Eight patients underwent endourological treatment and 10 patients open surgery including partial ureterectomy performed on 8 patient. The follow up period was 3 to 104 months (mean 37 months). Among those defined as imperative, the histopathological stage was pT1 in one, pT2 in one, pT3 in 3 and one in pT4. Among the elective cases, the histopathological stage was pTa in 7, pT1 in 2, pT2 in one, pT3 in 2 patients. Of the three defired as elective with tumors cT2 or higher, two died of disease. The 5-year survival rate was 50% and 68% in the imperative and elective cases, respectively. In the patients with tumors pT2 or higher and/or grade 3, the prognosis was poor which suggests the need for intensive therapy including lymph node dissection and/or adjuvant chemotherapy. It is necessary to consider the possibility of selecting nephron-sparing surgery for locally advanced tumors.

[Squamous cell carcinoma of the bladder presenting with bladder rupture: a case report].

A 71-year-old woman with pan-peritonitis was transferred to our hospital. Imaging studies showed the extravasation of contrast medium from the bladder to the intraperitoneal cavity, the right non-visualizing kidney and the left hydronephrosis. Cystoscopy revealed bladder stones and a fistula at the posterior wall of the bladder. A histological examination of specimen around the fistula revealed squamous cell carcinoma(SCC) with stromal invasion. Symptoms of peritonitis subsided following the placement of bilateral ureteral catheters and a urethral catheter. Because a laparotomy revealed peritonitis-carcinomatosa, she underwent bilateral ureterocutaneostomies, the removal of the bladder stones and the primary closure of the fistula. She died on the 42nd day after surgery.

Constitutive activation of the 41- and 43-kDa mitogen-activated protein (MAP) kinases in the progression of prostate cancer to an androgen-independent state.

AIM: The 41- and 43-kDa mitogen-activated protein kinases (MAPK; ERK2 and ERK1, respectively) play pivotal roles in the mitogenic signal transduction pathway. We previously demonstrated that constitutive activation of the MAPK cascade was related to the carcinogenesis of human tumors. In this study, we examined whether constitutive activation of MAPK was related to the progression to androgen independence of prostate cancer. METHODS: MAPK activation was examined by the appearance of phosphorylated forms and an in vitro kinase assay in four human (androgen-dependent and independent) prostate cancer cell lines and rat prostate cancer cell line Dunning (androgen-sensitive G line, and androgen-independent AT-3, AT-6 sublines). In addition, when androgen-dependent mouse Shionogi Carcinoma 115 (SC115) cells were serially cultured without androgen to obtain androgen-independent cells, the time and degree of MAPK activation were examined. RESULTS: One of three human androgen-independent cell lines (DU145) showed constitutive activation of MAPK, while an androgen-dependent cell line (LNCaP) did not show MAPK activation. While MAPK were not activated in an androgen-sensitive Dunning G cell line, MAPK were activated in androgen-independent sublines (AT-3 and AT-6) derived from a G cell line. In addition, when SC115 cells were serially cultured without androgen, the cells 16-24 weeks after androgen removal showed MAPK activation. Furthermore, in subcloned cells, MAPK activation was observed even in the cells maintained for 9 weeks in medium without testosterone. CONCLUSIONS: The present fi ndings suggest that constitutive activation of MAPK may be associated with the acquisition of hormone independence in prostate cancer and that clonal selection after androgen removal and hormone-independent growth through the MAPK signal transduction pathway could begin at a relatively early period in the individual cells.

[Laparoscopic radical nephrectomy for renal cell carcinoma at Kobe City General Hospital].

Between April 2002 and March 2004, 21 patients with clinical T1-T3a renal cell carcinoma underwent laparoscopic radical nephrectomy at the Kobe City General Hospital. A transperitoneal approach was chosen in 9 patients with clinical T2 stage, tumors more than 5 cm in diameter at the upper pole of the kidney, and posteriorly protruded tumors at the middle portion. Otherwise, a retroperitoneal approach was chosen in 12 patients. The mean (range) operative time for the transperitonal and retroperitoneal approaches was 355 (290-410) min and 342 (275-490) min, respectively. There were no major intraoperative complications. Postoperatively, one patient underwent emergency operation due to the perforation of duodenal ulcer. In the mean follow-up of 15.9 months, lung metastasis was seen in one patient under adjuvant immunotherapy. Significant differences between transperitoneal and retroperitoneal approaches were seen in mean time to renal artery clipping (170 versus 85 min, p < 0.01), mean blood loss (548 versus 281 ml, p < 0.05) and concurrent adrenalectomy (66.7 versus 16.7%, p < 0.05). Laparoscopic radical nephrectomy is a safe and feasible procedure when suitable approaches are chosen depending on tumor size and location.

[Retroperitoneoscopic partial nephrectomy for renal cell carcinoma: our initial experience and review of the literature].

PURPOSE: We report our initial experiences of retroperitoneoscopic partial nephrectomy for renal cell carcinoma. MATERIALS AND METHODS: From April 2002 to October 2003, we performed 11 retroperitoneoscopic partial nephrectomy for renal cell carcinoma. Our indication was T1N0M0 renal tumors which sizes were about 4 cm or less and were exophytic and were not situated at the renal hilum. First we inserted single J catheter to the renal pelvis cystoscopically. under fluoroscopic guidance. Next 4 trocars were set at pneumoretroperitoneum and the renal artery and vein were clamped individually after cold saline was circulated from the single J catheter. Renal parenchyma was sharply cut with scissors and vessels were coagulated with bipolar coagulator. When renal collecting system was opened, cariceal suture repair was performed. We used the microwave tissue coagulator without clamping the renal pedicle when the tumor was 2 cm or less in diameter and the distance from the tumor edge to the renal collecting system was more than 1cm. RESULTS: In 9 cases renal pedicles were clamped and in 2 cases were not. Mean tumor size was 27.5 +/- 8.9 mm and mean operative time was 350 +/- 92 minutes and mean estimated blood loss was 743 +/- 998 ml, and mean warm ischemic time was 70 +/- 30 minutes. In one case bleeding from cut surface was uncontrollable, so open conversion was needed. In this case the renal artery and vein were clamed but another artery exited. The surgical margins were all negative, and no other complications were happened. Post operative serum creatinine raised soon after the operation but finally downed, and the mean up level was 0.07 ng/ml only. But RI examination revealed the residual renal damages were in proportioned to the warm ischemic times. During a mean followup of 8 months no patients has had local recurrence or metastatic disease. CONCLUSIONS: Retroperitoneoscopic partial nephrectomy for renal cell carcinoma is effective for select patients. But better cooling method and earlier suture technique and more long follow-up periods will be necessary for establishment.

Traumatic renal artery thrombosis with renovascular hypertension.

A 15-year-old boy, who tumbled from a fourth-floor window, was transported to our hospital. Enhanced computed tomography (CT) 1.5 h after the injury showed a non-contrasted right kidney, and a repeat CT 6 h after the injury showed a growing retroperitoneal hematoma. The angiography showed complete obstruction of the right renal artery and bleeding from the subcapsular artery, which was successfully embolized. Renovascular hypertension developed on the second day after the injury; therefore, simple nephrectomy was performed.

Micropapillary variant of transitional cell carcinoma of the bladder.

A 77-year-old man visited the Kobe City General Hospital complaining of macroscopic hematuria. A computed tomography scan found a bladder tumor with left iliac and para-aortic lymph node metastasis. Two courses of cisplatin, cyclophosphamide and doxorubicin chemotherapy resulted in a minimal response. Radical cystectomy and a retroperitoneal lymph node dissection with bilateral ureterocutaneostomy reconstruction were then performed. A pathological examination revealed a micropapillary variant of transitional cell carcinoma (Grade 3, pT1pN2M1). The patient died of pelvic recurrence 7 months after the initiation of chemotherapy. Peritonitis carcinomatosa and lung metastases were observed at autopsy.

[Change of serum adrenal androgens in prostatic cancer patients after bilateral orchidectomy or LHRH agonist treatment].

Bilateral orchidectomy (ORX) or administration of luteinizing hormone releasing hormone agonist (LHRH) for prostatic cancer patients causes suppression of testicular androgens. However, the suppression of adrenal androgens by these treatments is controversial. We measured serum concentrations of testosterone (T), 4-androstene-3, 17-dione (A-dione), dehydroepiandrosterone (DHEA), LH, follicle-stimulating hormone (FSH), adrenocorticotropic hormone (ACTH) and cortisol before and after 3-12 months of the first hormonal treatment in 17 prostatic cancer patients who had received ORX (8 cases) or LHRH (9 cases). ORX and LHRH decreased serum T to the castration level significantly (ORX: p < 0.001, LHRH: p < 0.0001). ORX increased serum LH and FSH significantly (LH: p < 0.001, FSH: p < 0.001), whereas LHRH decreased LH and FSH significantly (LH: p < 0.05, FSH: p < 0.05). Neither treatment caused any significant change in ACTH or cortisol. ORX and LHRH decreased the serum A-dione significantly (ORX: p < 0.01, LHRH: p < 0.001). LHRH decreased the serum DHEA significantly (p < 0.01), whereas ORX did not decrease serum DHEA. These data suggest that "medical" and "surgical" castration, especially LHRH agonist, may decrease not only testicular androgens but also adrenal androgens.

Predicting disease outcome of non-invasive transitional cell carcinoma of the urinary bladder using an artificial neural network model: results of patient follow-up for 15 years or longer.

BACKGROUND: Patients with non-invasive (Ta/T1) transitional cell carcinoma (TCC) of the urinary bladder are often observed without progression in the long-term follow-up period, although many of them experience recurrence of disease. It is difficult to accurately predict the disease outcome of each patient with Ta/T1 TCC using conventional prognostic criteria. In this study, we examined the usefulness of artificial neural networks (ANNs) to predict the long-term disease outcome of patients with TCC of the urinary bladder. METHODS: A retrospective, prognostic study of 90 patients with Ta/T1 TCC of the urinary bladder, diagnosed by transurethral resection of the bladder tumor between April 1981 and March 1985, and then followed up for 15 years or longer, was carried out. Data were analyzed using the Bayesian network tool of SPSS Neural Connection 2.1. The input neural data consisted of tumor stage, grade, tumor number, age, gender, tumor architecture and estimates of mean nuclear volume. The data set was randomly divided into 68 training and 22 testing examples for the prediction of disease progression and tumor recurrence within 15 years. RESULTS: During 15 years follow-up, tumor recurrence was noted in 42/90 (47%) Ta/T1 tumors. The ANN model could not predict tumor recurrence. Conversely, disease progression was noted in 17/90 (19%) Ta/T1 tumors, and, in the test set, 4/22 (18%) Ta/T1 tumors underwent disease progression. The sensitivity of the ANN model to predict progression was 100% (specificity 67%; positive predictive value 40%; negative predictive value 100%). Patients who were judged to have a favorable prognosis using ANN analysis did not progress within the 15-year follow-up period. CONCLUSION: The results of the ANN study indicate that long-term progression-free survival of patients with non-invasive TCC of the urinary bladder can be precisely predicted. A favorable prognosis using ANNs would be one of the exclusion criteria for immediate or future total cystectomy.

Ductal adenocarcinoma of the prostate associated with prostatic multilocular cyst.

A case is reported here of ductal adenocarcinoma of the prostate forming a multilocular cyst, which responded well to androgen deprivation therapy.

Application of the microwave tissue coagulator: is it beneficial to partial nephrectomy?

BACKGROUND/AIM: The indications of partial nephrectomy have expanded after the introduction of new techniques for preventing excessive blood loss and avoiding deterioration of the renal function after clamping the renal pedicle. We present our clinical experience of partial nephrectomy for renal tumors using a microwave tissue coagulator. PATIENTS AND METHODS: Between April 1996 and January 2000, 34 patients underwent open partial nephrectomies in the Kobe City General Hospital. The microwave tissue coagulator was used for resection of the renal parenchyma, but in deeper lesions a sharp dissection was performed. Twenty-two patients (groups 1 and 2) underwent partial nephrectomy without vascular control (14 renal pedicles were not disturbed in group 1 patients, and 8 renal pedicles were dissected but not clamped in group 2 patients). Another 12 patients (group 3) underwent vascular control with ligation of the tumor-feeding segmental arteries before parenchymal resection. The patients of group 1 underwent wedge resections, while those of groups 2 and 3 underwent segmental or transverse partial nephrectomies. RESULTS: Complete tumor resection was done in all 34 patients. In group 1, the microwave tissue coagulator was very effective to control the blood loss (mean 330 ml). In larger resections, this method only was inadequate to control the blood loss (mean 489 ml in group 2), so that we needed vascular control. However, despite vascular control, mean blood losses of about 943 ml because of deeper venous bleeding occurred in group 3, and, moreover, postoperative renal infarctions occurred in 2 patients. Other complications were urinary fistula formation in 16 patients (47%) and renal pelvic stenoses in 2 patients (5.8%). All of the urinary fistulas were easily repaired by simple suturing intraoperatively. CONCLUSIONS: Especially in wedge resection, the microwave tissue coagulator achieved safe resection without vascular control which differs from other new techniques. However, in larger resections, a combination with other techniques may be necessary to decrease blood loss and the rate of complications.