Pelargonium sidoides extract mediates nephrotoxicity through mitochondrial malfunction and cytoskeleton destabilization.

We investigated the cytotoxic effect of Pelargonium sidoides extract on Madin-Darby canine kidney (MDCK) cells. P. sidoides extract decreased the cell viability in a dose dependent manner (> 0.2%). The extract of P. sidoides decreased the mitochondrial action potential, increased the number of reactive oxygen species (ROS) inside the cell, and caused nicotinamide adenine dinucleotide hydride (NADH) to be released, all of which are signs of mitochondrial dysfunction. The results of unbiased mRNA sequencing showed that 0.3% P. sidoides extract upregulates the apoptosis-related gene (BBC3). This finding was supported by immunoblot analysis of apoptosis signal pathways, which included Bcl-2, Bax, cytochrome C (CytC), cleaved caspase 3 (CC3), cleaved caspase 7 (CC7), cleaved caspase 9 (CC9) and cleaved PARP (CP). It is interesting to note that the elevated levels of Bax, CytC, CC3, CC7, and CC9, as well as CP, were suppressed by N-acetyl-L-cysteine (NAC) pretreatment, which points to ROS-mediated apoptosis. The small GTPases, RhoA, and Rac1/cdc42-GTP-bound active form were all lowered when P. sidoides extract was used. Also, RhoA-related cytoskeleton signals (ROCK, p-LIMK1/2, p-cofilin) and Rac1/cdc42-related signals (N-WASP, WAVE-2) were inhibited by P. sidoides extract. NAC or RhoA/Rac1/cdc42 activator pretreatment reduced P. sidoides extract-induced actin destabilization. In this work, P. sidoides extract promotes apoptosis by causing mitochondrial dysfunction and cytoskeleton disassembly.

Regenerative Strategy of Gold Electrodes for Long-Term Reuse of Electrochemical Biosensors.

Gold is of considerable interest for electrochemical active surfaces because thiol-modified chemicals and biomolecules can be easily immobilized with a simple procedure. However, most gold surfaces are damaged with repetitive measurements, so they are difficult to reuse. Here we demonstrate a novel electrochemical cleaning method of gold surfaces to reuse electrodes with a simple protocol that is easy and nontoxic. This electrochemical cleaning consists of two steps by using different solutions. The 1st step is a cyclic voltammetry sweep using a very low concentration of sulfuric acid, and the 2nd step is a cyclic voltammetry sweep using potassium ferricyanide. Different cleaning methods were also considered for comparison. Consequently, after assembling and desorption of the cell and antigen, the changes in gold electrode performance, as immunosensor and cytosensor, were investigated by electrochemical impedance and cyclic voltammetry. It was found that repetitive measurement is possible until five times while maintaining the reproducibility. It is believed that this method is capable of enabling reuse of gold electrodes and can be used for long-term and accurate monitoring of biological effects, especially at a low cost.

Key factors to establish the ovalbumin-induced atopic dermatitis minipig model: age and body weight.

BACKGROUND: Given its similar structure and immune response to the human skin, porcine is a good model for dermal studies. Here, we sensitized ovalbumin (Ova) on minipig back skin for 2-4 weeks to induce chronic atopic dermatitis (AD). RESULTS: Gross observation, serum cytokine level, epidermal thickness, and epidermal integrity did not change after 4 weeks of Ova induction compared with the control, indicating AD modeling failure. Only the neutrophils in the blood and macrophages in bronchoalveolar lavage fluid changed slightly until 3 or 2 weeks after Ova sensitization, respectively. The successful and failed Ova-induced AD minipig models only differ in age and body weight of the minipigs. The minipigs, 12 months old with a 30-kg median weight, had a two-fold thicker dermis than minipigs 8-10 months old, with an 18.97-kg median weight, resulting in impaired Ova permeability and immune response. CONCLUSION: Age and body weight are key factors that should be considered when developing an Ova-induced AD minipig model.

Disposable electrocatalytic sensor for whole blood NADH monitoring.

Monitoring nicotinamide adenine dinucleotide (NADH) is important because NADH is involved in cellular redox reactions and cellular energy production. Currently, few biosensors quantify NADH in whole blood. However, they still have limitations due to several defects, including poor repeatability, long analysis time, and their requirement of extra sample pretreatment. In this study, we developed electrocatalytic sensors using screen-printed electrodes with a redox-active monolayer 4'-mercapto-N-phenylquinone diamine formed by a self-assembled monolayer of a 4-aminothiophenol (4-ATP). We exhibited their behavior as electrocatalysts toward the oxidation of NADH in whole blood. Finally, the electrocatalytic sensors maintained stability and exhibited 3.5 µM limit of detection, with 0.0076 ± 0.0006 µM/µA sensitivity in a mouse's whole blood. As proof of concept, a polyhexamethylene guanidine phosphate-treated mouse model was used to induce inflammatory and fibrotic responses, and NADH level was measured for 45 days. This work demonstrates the potential of electrocatalytic sensors to analyze NADH in whole blood and to be developed for extensive applications.

Establishment of patient-derived xenografts from patients with gastrointestinal stromal tumors: analysis of clinicopathological characteristics related to engraftment success.

Patient-derived xenografts (PDXs) can represent the heterogeneity and histological characteristics of tumors and are thus useful for testing the efficacy of anti-cancer drugs; however, PDXs are difficult to generate, especially for gastrointestinal stromal tumor (GIST). We analyzed the clinicopathologic factors associated with the successful establishment of GIST PDX in NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mice. We used 185 GIST tumor fragments from patients who underwent surgical resection prior to (n = 66; 35.7%) and after treatment (n = 119; 64.3%) with tyrosine kinase inhibitors. The overall success rate of PDX establishment was 17%; in univariate analysis, engraftment success was associated with after TKI treatment, larger tumor size, higher mitotic count, higher Ki-67 index, higher cellularity, presence of tumor necrosis, primary mutations in KIT exon 11, and originating from metastatic lesions. In multivariate analysis, higher Ki-67 index, after TKI treatment, and larger tumor size were independent factors for engraftment success. Immunohistochemistry in representative samples further corroborated the above results. These results will be useful in the establishment of PDX models from GISTs.

Treatment of peri-invagination lesion and vitality preservation in an immature type III dens invaginatus: a case report.

BACKGROUND: To report a case of type III dens invaginatus associated with peri-invagination periodontitis in an immature permanent mandibular central incisor with open apex, in which only the invagination area was treated and vitality was preserved. CASE PRESENTATION: A 9-year-old boy was referred complaining of pain in the mandibular left central incisor. After radiographic examination, an invagination into the pulp chamber of the tooth associated with periapical radiolucency was detected. Endodontic access was performed and the orifice was identified under a dental operating microscope. The invagination area was chemo-mechanically cleaned. After 1 week, the invagination was obturated with mineral trioxide aggregate. During the 2-year follow up period, the tooth was asymptomatic. Radiographic examination revealed significant progression of periapical healing and root development in the main root canal of the tooth. CONCLUSION: Non-surgical root canal treatment of the invagination may preserve pulp vitality, and continuous root development of the tooth.

Clinical significance of MET gene amplification in metastatic or locally advanced gastric cancer treated with first-line fluoropyrimidine and platinum combination chemotherapy.

OBJECTIVE: To investigate the clinical significance of MET gene amplification in patients with gastric cancer in the palliative setting. METHODS: MET amplification was assessed using fluorescence in situ hybridization (FISH) in 50 patients and quantitative polymerase chain reaction (qPCR) in 326 patients; 259 patients treated with first-line fluoropyrimidine and platinum were included for survival analysis. RESULTS: The results of FISH and qPCR indicated that the c-MET/CEP7 ratio was correlated with gene copy number. The optimal cutoff value for the copy number using qPCR to detect MET gene amplification with FISH was 5 (κ=0.778, P<0.001). Twenty-one out of 326 patients (6.4%) were identified asMET amplification with a copy number of >5 detected by qPCR. MET-amplified gastric cancer was associated with an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of ≥2 (33.3% vs. 10.5% P=0.007), peritoneal metastasis (76.2% vs. 46.2%, P=0.008), and elevated bilirubin levels (28.6% vs. 7.3%, P=0.006). The median overall survival (OS) and progression-free survival (PFS) were 11.9 and 5.6 months, respectively. MET-amplified gastric cancer was not associated with survival outcomes [hazard ratio (HR)=0.68, 95% confidence interval (95% CI): 0.35-1.32, P=0.254 for PFS; HR=0.68, 95% CI: 0.35-1.32, P=0.251 for OS]. CONCLUSIONS: qPCR can be used to detect MET gene amplification. MET amplification was not a predictor of poor prognosis in patients with metastatic or unresectable gastric cancer.

Efficacy and retrievability of root canal filling using calcium silicate-based and epoxy resin-based root canal sealers with matched obturation techniques.

This study aimed to compare the efficiency of root canal filling procedures and the retrievability of the filling material with various sealers. Forty-three patients assigned to endodontic treatment with (i) continuous wave of condensation technique (CTW) with AH-plus (ii) single-cone technique (SCT) with EndoSeal MTA. The spent time, voids entrapping and postoperative symptoms were evaluated. To evaluate the retrievability, mandibular premolar (n = 60) were divided into four groups: AH-plus/CTW, EndoSeal MTA/SCT, MTA Fillapex/SCT and EndoSequence BC Sealer/SCT. The time required removing the filled materials and remnant score were examined. EndoSeal MTA/SCT showed significantly shorter time of filling procedure. The number of void did not show significant differences between two techniques. No patients showed clinical signs during the follow-up periods. There were no significant differences between group AH-plus and EndoSeal MTA for remnant score. A certain calcium silicate-based sealer with SCT may give similar clinical efficiencies as much as continuous-wave technique using AH-plus sealer.

Measuring Bone Biomarker Alkaline Phosphatase with Wafer-Scale Nanowell Array Electrodes.

Biosensors that can analyze a single drop of biological fluid can overcome limitations such as extraction volume from humans or animals, ethical problems, time, and cost. In this work, we have developed a highly sensitive electrochemical (EC) biosensor based on a nanowell array (NWA) for the detection of alkaline phosphatase (ALP), a serum indicator of bone formation. The size of the electrode is 2 × 1 mm2 and has over 10 million nanowells (400 nm diameter) arranged uniformly on the electrode surface. For detecting ALP, anti-ALP was immobilized and oriented on the NWA surface using a self-assembled monolayer and protein G. EC impedance spectroscopy (EIS) was used to determine the amount of ALP in 10 µL of sample. The impedance was calibrated with ALP concentration. The NWA has a linear dynamic range from 1 pg/mL to 100 ng/mL with a limit of detection (LOD) at 12 pg/mL. We used the sensor to measure the ALP in real mouse serum from 4, 10, and 20 weeks old mice and compared the results to the standard photometric assay. This work demonstrates the potential of EC NWA sensors to analyze a single drop of a real body fluid sample and to be developed for broad applications.

Innovations in biomedical nanoengineering: nanowell array biosensor.

Nanostructured biosensors have pioneered biomedical engineering by providing highly sensitive analyses of biomolecules. The nanowell array (NWA)-based biosensing platform is particularly innovative, where the small size of NWs within the array permits extremely profound sensing of a small quantity of biomolecules. Undoubtedly, the NWA geometry of a gently-sloped vertical wall is critical for selective docking of specific proteins without capillary resistances, and nanoprocessing has contributed to the fabrication of NWA electrodes on gold substrate such as molding process, e-beam lithography, and krypton-fluoride (KrF) stepper semiconductor method. The Lee group at the Mara Nanotech has established this NW-based biosensing technology during the past two decades by engineering highly sensitive electrochemical sensors and providing a broad range of detection methods from large molecules (e.g., cells or proteins) to small molecules (e.g., DNA and RNA). Nanosized gold dots in the NWA enhance the detection of electrochemical biosensing to the range of zeptomoles in precision against the complementary target DNA molecules. In this review, we discuss recent innovations in biomedical nanoengineering with a specific focus on novel NWA-based biosensors. We also describe our continuous efforts in achieving a label-free detection without non-specific binding while maintaining the activity and stability of immobilized biomolecules. This research can lay the foundation of a new platform for biomedical nanoengineering systems.

Establishment and characterization of patient-derived xenograft models of gastrointestinal stromal tumor resistant to standard tyrosine kinase inhibitors.

Gastrointestinal stromal tumors (GISTs) with KIT or platelet-derived growth factor receptor alpha (PDGFRa) oncogenic driver gene mutations, respond to tyrosine kinase inhibitors (TKIs) including imatinib, sunitinib, and regorafenib. However, most patients develop TKI resistance; therefore, novel agents are required. We established three TKI-resistant GIST patient-derived xenograft (PDX) models for effective drug development. These were PDX models harboring primary and secondary KIT and additional mutations; KIT exon 11 (p.Y570_L576del), KIT exon 17 (p.D816E), and PTEN (p.T321fs) mutations in GIST-RX1 from a patient who was unresponsive to imatinib, sunitinib, and sorafenib, and KIT exon 11 (p.K550_splice) and KIT exon 14 (p.T670I) mutations in GIST-RX2 and KIT exon 9 (p.502_503insYA) and KIT exon 17 (p.D820E) mutations in GIST-RX4 from patients with imatinib and imatinib/sunitinib resistance, respectively. The histological features and mutation statuses of GIST PDXs were consistent with those of the original patient tumors, and the models showed TKI sensitivity comparable to clinical responses. Imatinib inhibited the KIT pathway in imatinib-sensitive GIST-T1 but not GIST-RX1, RX2, and RX4. These GIST PDX models will be useful for studying TKI resistance mechanisms and evaluating novel targeted agents in GIST.

Polypyrrole Films with Micro/Nanosphere Shapes for Electrodes of High-Performance Supercapacitors.

We demonstrate a simple and efficient one-step procedure for synthesizing a solid state polypyrrole (PPy) thin film for supercapacitor applications using alternating current impedance spectroscopy. By controlling the frequency and amplitude we were able to create unique PPy nano/microstructures with a particular morphology of the loop. Our PPy micro/nanosphere shows extremely high capacitance of 568 F/g, which is close to the theoretical value of 620 F/g and 20-100% higher than that of other reported PPy electrodes. Most of all, this material presents high capacitance and significantly improved electrochemical stability without pulverization of its structure, demonstrating 77% retention of the capacitance value even after 10 000 charge/discharge cycles. These results are a consequence of the larger surface area and adequate porosity generated due to the balance between the nano/micro PPy loops. This created porous structure also allows the favored penetration of electrolyte and high ion mobility within the polymer and prevents the mechanical failure of the physical structure during volume variation associated with the insertion/deinsertion of ions upon cycling.

Single Microfluidic Electrochemical Sensor System for Simultaneous Multi-Pulmonary Hypertension Biomarker Analyses.

Miniaturized microfluidic biosensors have recently been advanced for portable point-of-care diagnostics by integrating lab-on-a-chip technology and electrochemical analysis. However, the design of a small, integrated, and reliable biosensor for multiple and simultaneous electrochemical analyses in a single device remains a challenge. Here, we present a simultaneous microfluidic electrochemical biosensing system to detect multiple biomarkers of pulmonary hypertension diseases in a single device. The miniaturized biosensor, which is composed of five chambers, is precisely and individually controlled using in-house-built pneumatic microvalves to manipulate the flow pathway. Each chamber is connected to an electrochemical sensor designed to detect four different biomarkers plus a reference control. Our design allows for loading of multiple reagents for simultaneous analyses. On the basis of the developed microfluidic electrochemical sensor system, we successfully detected four well-defined pulmonary hypertension-associated biomarkers, namely, fibrinogen, adiponectin, low-density lipoprotein, and 8-isoprostane. This novel approach offers a new platform for a rapid, miniaturized, and sensitive diagnostic sensor in a single device for various human diseases.

Nonmediated, Label-Free Based Detection of Cardiovascular Biomarker in a Biological Sample.

Direct electrochemical (EC) monitoring in a cell culture medium without electron transporter as called mediator is attractive topic in vitro organoid based on chip with frequently and long-time monitoring since it can avoid to its disadvantage as stability, toxicity. Here, direct monitoring with nonmediator is demonstrated based on impedance spectroscopy under the culture medium in order to overcome the limitation of mediator. The applicability of EC monitoring is shown by detecting alpha-1-anti trypsin (A1AT) which is known as biomarkers for cardiac damage and is widely chosen in organoid cardiac cell-based chip. The validity of presented EC monitoring is proved by observing signal processing and transduction in medium, mediator, medium-mediator complex. After the observation of electron behavior, A1AT as target analyte is immobilized on the electrode and detected using antibody-antigen interaction. As a result, the result indicates limit of detection is 10 ng mL-1 and linearity for the 10-1000 ng mL-1 range, with a sensitivity of 3980 nF (log [g mL])-1 retaining specificity. This EC monitoring is based on label-free and reagentless detection, will pave the way to use for continuous and simple monitoring of in vitro organoid platform.

Prognostic impact of fibroblast growth factor receptor 2 gene amplification in patients receiving fluoropyrimidine and platinum chemotherapy for metastatic and locally advanced unresectable gastric cancers.

Although Fibroblast growth factor receptor (FGFR) 2 gene amplification and its prognostic significance have been reported in resectable gastric cancers, information on these features remains limited in the metastatic setting. The presence of FGFR2 amplification was assessed in formalin-fixed, paraffin-embedded tissues using a quantitative PCR-based gene copy number assay with advanced gastric cancer cohorts. A total of 327 patients with tumor portion of ≥70% were analyzed for clinical features. Among these patients, 260 who received first-line fluoropyrimidine and platinum chemotherapy were analyzed for survival.Sixteen of 327 patients (4.9%) exhibited FGFR2 amplification. The amplification group showed associations with age <65 years, Borrmann type 4 disease, poor performance status, poorly differentiated histology, extra-abdominal lymph node metastases, and bone metastases. The median overall survival (OS) and progression-free survival (PFS) were found to be 12.7 and 5.8 months, respectively. In univariate analysis, PFS did not differ between amplification and no amplification groups (hazard ratio [HR]=1.34, 95% confidence interval [CI]: 0.78-2.31, p=0.290), although the OS was significantly shorter in the amplification group (HR=1.92, 95% CI: 1.13-3.26, p=0.015). However, multivariate analysis indicated that FGFR2 amplification was not an independent prognostic factor for OS (HR=1.42, 95% CI: 0.77-2.61, p=0.261).Although FGFR2 amplification is associated with poorer OS, it does not appear to be an independent prognostic predictor in patients with advanced gastric cancer treated with palliative fluoropyrimidine and platinum chemotherapy.

FGFR2 Assessment in Gastric Cancer Using Quantitative Real-Time Polymerase Chain Reaction, Fluorescent In Situ Hybridization, and Immunohistochemistry.

OBJECTIVES: Fibroblast growth factor receptor 2 (FGFR2) amplification has been reported to be a target for treatment in gastric cancer. However, an optimal tissue source and method for evaluating FGFR2 have yet to be established. METHODS: Copy numbers were compared by quantitative polymerase chain reaction (qPCR) using frozen vs formalin-fixed, paraffin-embedded (FFPE) tissue and biopsy vs surgical specimens. We correlated the results of qPCR and immunohistochemistry (IHC) with fluorescence in situ hybridization (FISH) using stage IV gastric cancer biopsy specimens and validated the results in surgical specimens. RESULTS: FFPE tissues were suitable for qPCR, and biopsy specimens were equivalent to or better than surgical specimens. qPCR and IHC results exhibited an excellent correlation with FISH at eight or more copies by qPCR in any kind of tissue, 5% or more by IHC in biopsy specimens, and 10% or more by IHC in surgical specimens. FGFR2 amplification was 6.6% in stage IV gastric cancers, and 42% of these showed heterogeneous amplification and overexpression. IHC indicated a good correlation with FISH even in the heterogeneous cases. CONCLUSIONS: FFPE biopsy tissues are an adequate source for FGFR2 evaluation in gastric carcinomas, and a qPCR-based copy number assay can be used for screening. IHC is also a valid and practical method for evaluating FGFR2, considering frequent heterogeneity.

Wafer-scale nanowell array patterning based electrochemical impedimetric immunosensor.

We have reported that nanowell array (NWA) can enhance electrochemical detection of molecular binding events by controlling the binding sites of the captured molecules. Using NWA biosensor based amperometric analysis, we have detected biological macromolecules such as DNA, protein or aptamers at low concentrations. In this research, we developed an impedimetric immunosensor based on wafer-scale NWA for electrochemical detection of stress-induced-phosphoprotein-1 (STIP-1). In order to develop NWA sensor through the cost-effective combination of high-throughput nanopattern, the NWA electrode was fabricated on Si wafer by krypton-fluoride (KrF) stepper semiconductor process. Finally, 12,500,000 ea nanowell with a 500 nm diameter was fabricated on 4 mm × 2 mm substrate. Next, by using these electrodes, we measured impedance to quantify antigen binding to the immunoaffinity layer. The limit of detection (LOD) of the NWA was improved about 100-fold compared to milli-sized electrodes (4 mm × 2 mm) without an NWA. These results suggest that wafer-scale NWA immunosensor will be useful for biosensing applications because their interface response is appropriate for detecting molecular binding events.

DZNep, inhibitor of S-adenosylhomocysteine hydrolase, down-regulates expression of SETDB1 H3K9me3 HMTase in human lung cancer cells.

3-Deazaneplanocin A (DZNep), an epigenetic anticancer drug, leads to the indirect suppression of S-adenosyl methionine-dependent cellular methylations by inhibiting S-adenosyl homocystein (AdoHcy) hydrolase. Although it is well known that DZNep targets the degradation of EZH2 protein, H3K27me3 HMTase, there are still uncertainties about the regulation of other types of HMTases during cell death. In this study, we describe that SETDB1 gene expression was regulated by DZNep treatment in human lung cancer cells. We confirm that DZNep induced growth inhibition and increased the dead cell population of lung cancer cells. DZNep treatment affected histone methylations, including H3K27me3 and H3K9me3, but not H3K4me3. Reduced levels of H3K27me3 and H3K9me3 were related with the decreased EZH2 and SETDB1 proteins. Real time PCR analysis showed that SETDB1 gene expression was decreased by DZNep treatment, but no effect was observed for EZH2 gene expression. We cloned the promoter region of SETDB1 and SUV39H1 genes, and performed luciferase assays. The promoter activity of SETDB1 gene was down regulated by DZNep treatment, whereas no effect on SUV39H1 promoter activity was observed. In conclusion, we suggest that DZNep regulates not only on H3K27me3 HMTase EZH2, but also H3K9 HMTase SETDB1 gene expression at the transcription level, implicating that the mechanism of action of DZNep targets multiple HMTases during the death of lung cancer cells.