In vitro throughput screening of anticancer drugs using patient-derived cell lines cultured on vascularized three-dimensional stromal tissues.

The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. STATEMENT OF SIGNIFICANCE: To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine.

Differential gene expression analysis using RNA sequencing: retinal pigment epithelial cells after exposure to continuous-wave and subthreshold micropulse laser.

PURPOSE: Subthreshold micropulse laser (SMPL) is more clinically efficient for the treatment of diabetic macular edema (DME) than the conventional continuous-wave (CW) laser. We aimed to characterize transcriptome changes after the application of these lasers and to compare the transcripts. METHODS: Human pluripotent stem cell-derived retinal pigment epithelial cells were exposed to laser irradiation. Differentially expressed genes (DEGs), distribution of heat shock protein (Hsp) family, gene expression profile, and gene ontology (GO) enrichment analysis based on RNA sequencing data were investigated at 3 h and 24 h after irradiation. RESULTS: CW laser induced more DEGs than SMPL (1771 vs. 520 genes). The expression of the Hsp family was confirmed in both groups: however, the induction patterns was different for different genes. GO enrichment analysis revealed that CW laser upregulated the expression of DEGs involved in vasculature development (GO: 0001944), related to apoptosis and repair after cell injury whereas SMPL upregulated the expression of DEGs involved in photoreceptor cell maintenance (GO: 0045494), photoreceptor cell development (GO: 0042461), and sensory perception of light stimuli (GO: 0050953). CONCLUSIONS: The results provide insights into the genetic responses and may contribute to the understanding of the molecular mechanisms of laser-induced thermal effects.

Constructing vascularized hepatic tissue by cell-assembled viscous tissue sedimentation method and its application for vascular toxicity assessment.

In vitro Construction of the liver sinusoidal structure using artificial tissue is an important but worthwhile challenge, particularly for assessing the risk of diseases such as sinusoidal obstruction syndrome (SOS). Current models are unsuitable for evaluating the toxicity because of lacking sinusoidal capillary. In this study, we developed a vascularized hepatic tissue (VHT) using a unique tissue engineering technique, the cell assembled viscous tissue by sedimentation (CAViTs) method. The "viscous bodies" created using the CAViTs method exhibited significant self-assembly within 6 h after seeding, promoting cell-cell interaction. The level of albumin secreted by the VHT was four times higher than that of 2D-coculture and maintained for 1 month. The gene expression pattern of the VHT was closer to that of total human liver, compared with the 2D system. Quantitative evaluations of the vascular structure of VHT treated with two typical SOS-inducing compounds, monocrotaline and retrorsine, revealed higher sensitivity (IC50 = 40.35 µM), 19.92 times higher than the cell-viability assay. Thus, VHT represents an innovative in vitro model that mimics the vessel network structure and could become a useful tool for the early screening of compounds associated with a risk of vascular toxicity. STATEMENT OF SIGNIFICANCE: Mimicking sinusoidal structures in in vitro liver model is important to consider from the perspective of predicting hepatotoxicity such like sinusoidal obstruction syndrome (SOS). However, it was difficult to reconstruct the vascular structure within the hepatocyte-rich environment. In this study, we constructed a vascularized hepatic tissue in a high-throughput manner by a unique method using collagen and heparin, and evaluated its applicability to toxicity assessment. Vessel morphology analysis of the model treated by monocrotaline, which is a well-known SOS-inducing compound, could predict the toxicity with higher sensitivity. To the best of our knowledge, this is the first report to provide vascularized hepatic tissues using sinusoidal endothelial cells at least for demonstrating applicability to the evaluation of SOS induction risk.

Ultimate suppression of thermal transport in amorphous silicon nitride by phononic nanostructure.

Engineering the thermal conductivity of amorphous materials is highly essential for the thermal management of future electronic devices. Here, we demonstrate the impact of ultrafine nanostructuring on the thermal conductivity reduction of amorphous silicon nitride (a-Si3N4) thin films, in which the thermal transport is inherently impeded by the atomic disorders. Ultrafine nanostructuring with feature sizes below 20 nm allows us to fully suppress contribution of the propagating vibrational modes (propagons), leaving only the diffusive vibrational modes (diffusons) to contribute to thermal transport in a-Si3N4 A combination of the phonon-gas kinetics model and the Allen-Feldmann theory reproduced the measured results without any fitting parameters. The thermal conductivity reduction was explained as extremely strong diffusive boundary scattering of both propagons and diffusons. These findings give rise to substantial tunability of thermal conductivity of amorphous materials, which enables us to provide better thermal solutions in microelectronic devices.

Laterally Movable Triple Electrodes Actuator toward Low Voltage and Fast Response RF-MEMS Switches.

A novel actuator toward a low voltage actuation and fast response in RF-MEMS (radio frequency micro-electro-mechanical systems) switches is reported in this paper. The switch is comprised of laterally movable triple electrodes, which are bistable by electrostatic forces applied for not only the on-state, but also the off-state. The bistable triple electrodes enable the implementation of capacitive series and shunt type switches on a single switch, which leads to high isolation in spite of the small gap between the electrodes on the series switch. These features of the actuator are effective for a low voltage and fast response actuation in both the on- and off-state. The structure was designed in RF from a mechanical point of view. The laterally movable electrodes were achieved using a simple, low-cost two-mask process with 2.0 µm thick sputtered aluminum. The characteristics of switching response time and actuation voltage were 5.0 µs and 9.0 V, respectively.

Electrostatic MEMS Vibration Energy Harvesters inside of Tire Treads.

An electret electrostatic MEMS vibration energy harvester for tire sensors mounted inside of the tire tread is reported. The device was designed so as to linearly change an electrostatic capacitance between the corrugated electret and output electrode according to the displacement of the proof mass. The electromechanical linearity was effective at reducing the power loss. The output power reached 495 µW under sinusoidal vibration despite the footprint size being as small as 1 cm². Under impact vibration inside of the tire tread, the output power reached 60 µW at a traveling speed of 60 km/h. It was revealed that a higher mechanical resonance frequency of the harvester adjusted within the frequency band of a low-power spectral density of impact vibration acceleration was effective for high efficiency harvest impact vibration energy.

Importance of continuous sequential chemotherapy and multimodal treatment for advanced testicular cancer: a high-volume Japanese center experience.

Patients with "difficult-to-treat" advanced testicular cancer can require multiple therapies. We retrospectively assessed our patients with advanced germ cell tumors (GCTs) and characterized the clinical efficacy, outcomes, and factors affecting overall survival (OS).Two hundred fifty-three patients with advanced GCTs were treated at Kyoto Prefectural University of Medicine, Kyoto, Japan, from June 1998 to September 2013. Of 253 patients, 142 patients had salvage chemotherapy.As first-line therapy, bleomycin, etoposide, and cisplatin, and etoposide and cisplatin therapies were performed in 234 cases (92.5%). As second-line therapy, etoposide, ifosfamide, and cisplatin/vinblastine, ifosfamide, and cisplatin, and paclitaxel, ifosfamide, and cisplatin/paclitaxel, ifosfamide, and nedaplatin therapies were carried out in 44 and 59 cases, respectively. Furthermore, 111, 72, 44, and 28 cases had third, fourth, fifth, and sixth-or-later-line chemotherapy, respectively. Five-year OS rate stratified by chemotherapy line was 95.5% in the first line, 89.4% in the second line, 82.1% in the third line, 45.1% in the fourth line, and 58.9% in the fifth or after line. A statistical significant difference was found when comparing fourth-or-after-line versus first to third-line therapy. Additional procedures were performed, including retroperitoneal lymph node dissection (RPLND) (n = 168), extra-RPLN resection (n= 114), and external beam radiotherapy/stereotactic radiotherapy (n = 78).Multivariate analysis showed that factors predicting better outcomes were in serum tumor marker (STM) normalization, RPLND, and extra-RPLN resection.Good outcomes were obtained in patients who completed chemotherapy up to third line. After fourth-line chemotherapy, approximately 50% of "difficult-to-treat" patients could be cured with normalization of STM levels and residual mass resection. Continuous or sequential chemotherapy with multimodality therapy is important for patients with "difficult-to-treat" advanced GCTs. Effective chemotherapy after third line should be developed.

Initial experience of combined use of photodynamic diagnosis and narrow band imaging for detection of flat urothelial lesion.

BACKGROUND: This preliminary study is the first report to compare photodynamic diagnosis (PDD) with narrow band imaging (NBI) in the same patients with flat urothelial lesions suspicious of carcinoma in situ (CIS) of the bladder. METHODS: Between November 26, 2012 and April 1, 2013, 10 patients underwent transurethral resection of bladder tumor using PDD and NBI simultaneously because of suspicion of CIS. The bladder was mapped first under white light (WL), then under NBI, and subsequently under blue light in odd-numbered patients. The bladder was mapped first under WL, then under blue light, and subsequently under NBI in even-numbered patients. Biopsies were carried out from all suspicious areas, noting whether NBI, PDD or both detected lesions. Random cold cup biopsies from healthy mucosa of bladder were performed from lesions negative on PDD and NBI. RESULTS: The sensitivity and specificity of PDD for detection of CIS and dysplasia were 0.916 and 0.827, respectively. The sensitivity and specificity of NBI for detection of CIS and dysplasia were 0.625 and 0.879. The area under the curve (AUC) for detection of CIS and dysplasia was 0.872 with PDD and 0.752 with NBI. The AUC with combined use of PDD and NBI was 0.918. There was no cancer or dysplasia identified in 43 lesions that were both PDD- and NBI-negative. CONCLUSION: When both PDD and NBI are negative, the possibility of CIS or dysplasia might be very low. The usefulness of the combination of PDD with NBI was suggested in this study.

Comparison of diameter-axial-polar nephrometry and RENAL nephrometry score for treatment decision-making in patients with small renal mass.

INTRODUCTION: The aim of this study was to evaluate our institution's experience in performing laparoscopic radical nephrectomy (LRN) and partial nephrectomy (PN) in patients with small renal masses. METHODS: 142 patients with cT1aN0M0 lesions were identified. 68 of these subjects were treated with LRN and 74 were treated with laparoscopic PN (LPN). The clinicopathological characteristics of the two groups of patients, including diameter-axial-polar (DAP) nephrometry and RENAL nephrometry score (RENAL-NS), operative results, and outcomes, were retrospectively analyzed. RESULTS: A multivariate logistic regression analysis for the selection of PN as the treatment showed that tumor size, DAP nephrometry, RENAL-NS and imperative condition were all independent factors. The area under the curve receiver operating characteristics (ROC-AUC) of DAP and RENAL-NS for performing LPN were 0.897 and 0.825, respectively. CONCLUSIONS: Although LRN was performed in patients with a high nephrometry score in this study, open partial nephrectomy (OPN) should be considered for patients with a high nephrometry score in T1a renal cell carcinoma (RCC) because of better functional and similar oncological outcomes. Based on ROC analysis, when DAP is 6 or less, LPN should be considered and when DAP is 7 or more, OPN should be considered.

Magnetically promoted rapid immunoreactions using functionalized fluorescent magnetic beads: a proof of principle.

BACKGROUND: Accurate detection and monitoring of disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymer-coated fluorescent ferrite (FF) beads containing both ferrites (magnetic iron oxide) and fluorescent europium complexes. METHODS: FF beads were prepared by encapsulation of hydrophobic europium complexes into the polymer layers of affinity magnetic beads using organic solvent. A sandwich immunoassay using magnetic collection of antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specific antigen were selected as target detection antigens to demonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of antibody-coated FF beads onto carcinoma cell samples was also performed. RESULTS: The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF beads, detected target antigens within 5 min of sample addition. Without magnetic collection, the sandwich immunoassay using antibody-coated FF beads required long times, similar to conventional immunoassays. Using the magnetic collection of antibody-coated FF beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min. CONCLUSIONS: This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody-coated FF beads allow acceleration of the antigen-antibody reaction. The simple magnetic collection of antibody-coated FF beads to a specific space enables rapid detection of disease-related biomarkers and identification of carcinoma cells.

Retroperitoneoscopic heminephroureterectomy for children with duplex anomaly: Initial experience.

OBJECTIVES: To evaluate the feasibility of retroperitoneoscopic heminephroureterectomy for children with duplex anomaly. METHODS: Retroperitoneoscopic heminephroureterectomy was performed in five children (four girls and one boy) with complete duplication of the ureter, of whom four (age range 1-5 years; mean age 3.3 years) had upper pole ectopic megaureters and one (3 years old) had an upper pole megaureter with ureterocele. In the patient with ureterocele, distal ureterectomy and ureterocelectomy were performed by Pfannenstiel incision. RESULTS: The mean operation time was 346 min (range 270-450 min) in the four patients with ectopic megaureter and 420 min (330 min for heminephroureterectomy) in the patient with ureterocele. The mean estimated blood loss was 43 mL (range 5-100 mL) in the four patients with ectopic megaureter and 40 mL in the patient with ureterocele. No postoperative complications were observed. Postoperative intravenous pyelography showed normal pyelogram and renal function of the preserved lower pole in all cases. CONCLUSIONS: Retroperitoneoscopic heminephroureterectomy for children is feasible, safe and has good postoperative results, including cosmetic results. However, the operation time needs to be reduced.

A high-speed atomic force microscope for studying biological macromolecules in action.

The atomic force microscope (AFM), which was invented by Binnig et al. in 1986, can image at nanometer resolution individual biological macromolecules on a substrate in solution. This unique capability awoke an expectation of imaging processes occurring in biological macromolecules at work. However, this expectation was not met, because the imaging rate with available AFMs was too low to capture biological processes. This expectation has at last been realized by the high-speed AFM developed by our research group at Kanazawa University. In this article, after a brief review of the development of our apparatus, its recent advancement and imaging data obtained with motor proteins are presented.

Bladder capacity at the time of enuresis.

OBJECTIVES: To evaluate bladder capacity at the time of enuresis and compare the daytime capacity of enuretics with that of controls. METHODS: The functional bladder capacity (FBC), which was the maximal endurable capacity in the daytime, the ordinary bladder capacity (OBC), which was the capacity at ordinary urinary sensation in the daytime, and the enuretic bladder capacity (EBC), which was the capacity at the time of enuresis, were measured in 67 patients with monosymptomatic nocturnal enuresis. The EBC was measured using a diaper and enuresis alarm. The FBC, OBC, and nocturnal bladder capacity, which was the maximal voided volume in the nighttime, were also measured in 67 controls. RESULTS: The FBC was not significantly different between enuretics and controls. The EBC in enuretics was significantly smaller than the nocturnal bladder capacity in controls. In enuretics, the EBC was significantly smaller than the FBC and was similar to the OBC. In controls, no statistically significant differences were found between the FBC and the nocturnal bladder capacity. CONCLUSIONS: In enuretics, the bladder capacity during sleep was significantly smaller than the daytime functional capacity. In controls, on the other hand, the bladder capacity during sleep did not differ from the daytime functional capacity. Our understanding of the bladder capacity in enuretics may need to change in view of this result. The inability to hold urine during sleep may be an important cause of nocturnal enuresis.

Relationships among nocturnal urinary volume, bladder capacity, and nocturia with and without water load in nonenuretic children.

OBJECTIVES: To evaluate the relationships among nocturnal urinary volume (NUV), bladder capacity, and nocturia in nonenuretic children with and without water load. METHODS: We evaluated 72 healthy nonenuretic volunteers. The NUV and bladder capacity were measured for 2 nights at volunteers' homes with and without water load. RESULTS: Of the 72 nonenuretic children, 45 (63%) were classified into the large NUV group in which the NUV with water load was more than the upper limit of the 95% confidence interval of NUV in Japanese nonenuretic children, and the other 27 (37%) were classified into the small NUV group. In the large NUV group, 33 (73%) had one or two episodes of nocturia because of water intake, and 12 (27%) did not. The nocturnal bladder capacity in the latter 12 children was significantly larger than that in the former 33, although no significant differences were found in functional bladder capacity and NUV with water intake. CONCLUSIONS: Most nonenuretic children were able to cope with the physiologic loading of water that would cause bedwetting in enuretic children. After water loading, three quarters of the nonenuretic children were able to awake easily because of the need to urinate with a certain volume of urine in the bladder, and the one quarter who did not awake were able to hold more urine in their bladder in the sleep state.