Proteomic Analysis of Urothelium of Rats with Detrusor Overactivity Induced by Bladder Outlet Obstruction.

Overactive bladder (OAB) syndrome is a condition that has four symptoms: urgency, urinary frequency, nocturia, and urge incontinence and negatively affects a patient's life. Recently, it is considered that the urinary bladder urothelium is closely linked to pathogenesis of OAB. However, the mechanisms of pathogenesis of OAB at the molecular level remain poorly understood, mainly because of lack of modern molecular analysis. The goal of this study is to identify a potential target protein that could act as a predictive factor for effective diagnosis and aid in the development of therapeutic strategies for the treatment of OAB syndrome. We produced OAB in a rat model and performed the first proteomic analysis on the mucosal layer (urothelium) of the bladders of sham control and OAB rats. The resulting data revealed the differential expression of 355 proteins in the bladder urothelium of OAB rats compared with sham subjects. Signaling pathway analysis revealed that the differentially expressed proteins were mainly involved in the inflammatory response and apoptosis. Our findings suggest a new target for accurate diagnosis of OAB that can provide essential information for the development of drug treatment strategies as well as establish criteria for screening patients in the clinical environment.

Early stage high-content HIV diagnosis based on concurrent monitoring of actin cytoskeleton, CD3, CD4, and CD8.

Antiretroviral treatment can reduce the death rate of human immunodeficiency virus (HIV) infection, and its effectiveness is maximized at the early stage of HIV infection. The present study demonstrates an early stage high-content HIV diagnosis based on multicolor concurrent monitoring of CD4, CD8, and CD3 coreceptors and F-actin cytoskeleton using quantum dot (Qdot)-antibody conjugates at the single cell level. Artificial HIV infection of peripheral blood mononuclear cells (PBMCs) was achieved by the treatment of PBMCs with gp120 glycoproteins. Using the present system, it was determined that the CD4/CD8 ratios of normal PBMCs obtained from the blood samples of 11 adults were in the range of 1.04 to 1.52 as a result of the quantitative counting of single PBMCs while the CD4/CD8 ratios of artificial HIV-infected PBMCs were from 0.045 to 0.63. In addition, the structural changes of actin filament alignments in PBMCs bound to gp120 proteins were clearly observed by the multicolor single cell imaging system. This approach suggests a new model of accurate early stage HIV diagnosis simultaneously providing information on actin cytoskeleton and subtypes of PBMCs as well as their CD4/CD8 ratios.

Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration.

Although human lysyl-tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67-kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi-tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site-directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4-mediated ubiquitination, and KRS inhibited ubiquitin-dependent degradation of 67LR, thereby enhancing laminin-induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis.

VEGF inhibitor (Iressa) arrests histone deacetylase expression: single-cell cotransfection imaging cytometry for multi-target-multi-drug analysis.

Multi-target-multi-drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single-cell cotransfection imaging cytometry to quantitatively screen drug-induced off-target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP-HDAC/YFP-VEGF). These gene constructs were cotransfected in HEK-293 cells to explore the possibility of off-target effects of 4-phenylbutyrate and Iressa on the expression of VEGF and HDAC through single-cell imaging cytometry. Iressa (10 µM) treatment at the time of cotransfection or 48 h after cotransfection of RFP-HDAC/YFP-VEGF plasmids in HEK-293 cells resulted in off-target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4-Phenylbutyrate (2.0 mM) did not show any off-target effects on VEGF expression. The developed quantitative multicolor live single-cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi-target-multi-drug analysis by increasing the on-target/desired off-target effects and eliminating the undesirable off-target effects.

Synthesis of highly antibacterial nanocrystalline trivalent silver polydiguanide.

Highly monodispersed nanoparticles of a trivalent silver polydiguanide complex are synthesized by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with a polydiguanide ligand in a reverse microemulsion at room temperature. The synthesized nanoparticles have excellent photostability and displayed superior antibacterial activity toward Gram-positive and Gram-negative prokaryotes of clinical interest in vitro compared to silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Interdigitated microelectrode array-coupled bipolar semiconductor photodiode array (IMEA-PDA) microchip for on-chip electrochemiluminescence detection.

This paper reports the design, fabrication and testing of a microchip wherein interdigitated microelectrode arrays (IMEA) were integrated with bipolar semiconductor photodiode array (PDA) chip to fabricate a highly compact embodiment for on-chip handling of solutions and electrochemiluminescence (ECL) detection. A 12 x 12 micro array of photodiodes, each coupled with an interdigitated microelectrode array (IMEA), an array of current amplifiers, and a photodiode element-addressing circuit were integrated into a single 2 x 2 cm² IC chip. Each photodiode had dimensions of 300 x 300 µm² and the photodiode-to-photodiode distance was 100 µm. The chip was successfully applied to the on-chip quantification of electro-chemiluminescing probe-labeled single stranded oligonucleotides. The minimum detectable limit at signal/noise ≥ 3 was found to be 5 x 10⁻¹4 moles of oligonucleotides with a sample volume as low as 5 microl (i.e., 10 fmole/µl). The attractive features of the developed IMEA-PDA microchip are that a plurality of samples can be analyzed simultaneously using a chip and that for a given sample the data can be averaged from values obtained from multiple, individually addressed pixels. These in turn bring in speed and statistical confidence in analysis. The IMEA-PDA microchip system has the potential to be used as a versatile and highly compact chemical analysis tool for chemical sensing and metrology applications.

Nanocrystalline silver supported on activated carbon matrix from hydrosol: antibacterial mechanism under prolonged incubation conditions.

Nanocrystalline Silver-supported activated carbon (AC) was fabricated by directly loading silver nanoparticles into the porous AC matrix from a preformed nanosilver hydrosol. Silver-AC composites were also synthesized using a conventional thermal impregnation method. While XRD calculation indicated the presence of Ag crystallites in nanometer range, silver nanoparticle hydrosol-treated AC having the finest crystallite size CS (< 14.4 nm), SEM images clearly revealed that Ag crystals coalesced significantly with increasing temperature resulting in much larger particle size in thermally impregnated silver-AC composities. To clarify the antibacterial mechanism of silver nanoparticles impregnated into AC under prolonged incubation conditions the antibacterial activity was investigated against Gram-negative Escherichia coli. The kinetics of bacterial inactivation, in presence of hydroxyl radical (*OH) scavengers, and superoxide anion radical (*O2-) inducer suggest the contribution of the reactive oxygen species (ROS) to antibacterial effect. However, these ROS scavengers did not show any inhibition of bactericidal activity after approximately 1 h, suggesting that generated ROS are responsible for E. coli inactivation only during the initial 1 h of the incubation time. This study clearly indicates the plausible implication of eluted Ag+ as major lethal species responsible for the E. coli inactivation over extended process time. The antibacterial process was found to be highly promoted at higher temperature which was ascribed to the enhanced ROS formation and Ag+ elution at higher temperature. SEM images revealed considerable differences in the morphology of E. coli cells contacting with the virgin AC and that contacting with silver-supported AC. The strong antibacterial ability of formaldehyde-modified silver-supported AC further provided the indirect evidences for catalytic oxidation by ROS, and for the synergistic antibacterial effects of nanocrystalline silver and adsorbed formaldehyde. Comparison of the antibacterial activities of the silver-supported materials prepared by silver colloid deposition and by conventional thermal impregnation technique indicates that former is more efficient in controlling microorganism.

Green fluorescent protein (GFP) as a direct biosensor for mutation detection: elimination of false-negative errors in target gene expression.

A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(+/-)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10-500 microM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0-8.59 microM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE-AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.

Bifunctional Therapeutic High-Valence Silver-Pyridoxine Nanoparticles with Proliferative and Antibacterial Wound-Healing Activities.

The antibacterial and moisturizing effects inherent to silver nanoparticles contribute greatly to their use as a topical antibacterial agent. The antibacterial property of silver nanoparticles provides topical wounds with an indirect environment for healing through the prevention of pathogenic infection. However, the direct wound-healing effects of silver nanoparticles have not been previously explored. In this work, we report a bimodal therapeutic silver nanoparticle that possesses both direct wound-healing and antibacterial properties. The nanoparticles consist of high-valence silver-pyridoxine complexes. The wound-healing efficacy was verified in diabetic mice, as well as in vitro assays. A MAPK pathway study demonstrated that silver-pyridoxine nanoparticles induced the proliferation and migration of keratinocyte and fibroblast cells. Antibacterial activities in 8 different pathogenic bacteria responsible for the infection of burn wounds were tested. The rapid wound healing occurring on skin wounds of diabetic mice attests to the utility of bimodal therapeutic silver nanoparticles as a next-generation topical therapeutic agent.

Shape-Dependent Skin Penetration of Silver Nanoparticles: Does It Really Matter?

Advancements in nano-structured materials have facilitated several applications of nanoparticles (NPs). Skin penetration of NPs is a crucial factor for designing suitable topical antibacterial agents with low systemic toxicity. Available reports focus on size-dependent skin penetration of NPs, mainly through follicular pathways. Herein, for the first time, we demonstrate a proof-of-concept study that entails variations in skin permeability and diffusion coefficients, penetration rates and depth-of-penetration of differently shaped silver NPs (AgNPs) via intercellular pathways using both in vitro and in vivo models. The antimicrobial activity of AgNPs is known. Different shapes of AgNPs may exhibit diverse antimicrobial activities and skin penetration capabilities depending upon their active metallic facets. Consideration of the shape dependency of AgNPs in antimicrobial formulations could help developing an ideal topical agent with the highest efficacy and low systemic toxicity.

Real-time concurrent monitoring of apoptosis, cytosolic calcium, and mitochondria permeability transition for hypermulticolor high-content screening of drug-induced mitochondrial dysfunction-mediated hepatotoxicity.

A quantitative high-content screening (HCS) was suggested for the real-time monitoring of drug-induced mitochondrial dysfunction-mediated hepatotoxicity. This HCS is very advantageous in that it allows simultaneous observation of drug-induced activations of hepatotoxic pathways using hypermulticolor cellular imaging. The mitochondrial permeability transition (MPT), cytosolic calcium, and caspase-3 were selected as functional markers to verify drug-induced hepatotoxicity and were concurrently monitored in HepG2 cells in a real-time manner. Nefazodone, tolcapone, and troglitazone caused mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase. On the other hand, extrinsic pathway-mediated apoptotic cell death was monitored when HepG2 cells were treated with piroxicam. It was found that piroxicam-treated HepG2 cells showed apoptotic cell death without the MPT formation, while a cytosolic calcium increase was clearly observed. This finding was confirmed by the caspase-8 inhibition assay. These results demonstrated the unique potential of real-time hypermulticolor HCS to screen hepatotoxic drugs at the in vitro stage rather than the later in vivo stage based on an animal model and to ultimately reduce the probability of drug failure.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA.

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Knock-down of argonaute 2 (AGO2) induces apoptosis in myeloid leukaemia cells and inhibits siRNA-mediated silencing of transfected oncogenes in HEK-293 cells.

Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer.

Determination of UV-induced DNA damages to suppress protein expression using reporter gene assay-based single cell cotransfection imaging cytometry.

Effects of UVB or UVC-induced DNA damage were simultaneously monitored at single cellular level by analyzing the change of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) expression in human embryonic kidney (HEK) 293 cells using multicolor single-cell imaging cytometry. The method is based on the idea that there exists a quantitative correlation between the degree of UV-induced DNA damage and protein expression. A cotransfection assay was performed using UVB irradiated YFP and UVC irradiated RFP genes to eliminate cell-to-cell variation in protein expression yield. Up to an UVB irradiation dose of 50kJ/m(2), YFP expression yield did not change compared to control. On the other hand, RFP expression yield decreased remarkably as the UVC dose increased from 79.5 to 159J/m(2). The results showed that a certain level of DNA damage is efficiently repaired by intracellular repair mechanism and does not influence protein mutation. In addition, it was found that the amount of DNA damage induced by UVB in sunlight would not interfere with normal protein expression in the human body. Single-cell imaging cytometry is a cell lysis-free approach to directly monitor the intracellular correlation between the degree of UV-induced DNA damage and protein expression.

Simultaneous quantitative monitoring of drug-induced caspase cascade pathways in carcinoma cells.

Caspases are the key mediators of apoptosis. The caspase cascade includes a series of events leading to the activation of initiator and downstream caspases in a cell. Analysis of the caspase cascade in intact cells, however, has generally been limited as the simultaneous monitoring of upstream and downstream caspases is not well executed. In an effort to monitor the activation of caspase cascades in an intact cell, high-content cellular imaging that allows simultaneous quantitative monitoring of caspase activation has been developed. This has great significance for the exploration of various cellular caspases involved in apoptotic pathways as possible therapeutic targets in the process of drug discovery. To explore the potential of simultaneous monitoring of caspase-mediated apoptotic pathways, human myeloid leukemia HL-60 cells were treated with SH-03 {(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13a-tetrahydro-3H-chromeno [3,4-b]pyrano[2,3-h]chromen-7-ol} (a newly synthesized candidate), camptothecin or naringenin (agents known to induce apoptosis) with or without caspase inhibitors. SH-03 or naringenin treatment initiated the caspase cascade through an intrinsic apoptotic pathway, whereas camptothecin treatment triggered both intrinsic and extrinsic caspase cascades. We now report a new approach based on uniform threshold intensity distribution that facilitates rapid, quantitative monitoring of drug-induced caspase cascades through multi-spectral and multicolor imaging cytometry.

Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli.

In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag(+) (in the form of AgNO(3)). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.

Determination of the dose-depth distribution of proton beam using resazurin assay in vitro and diode laser-induced fluorescence detection.

In this study the dose-depth distribution pattern of proton beams was investigated by inactivation of human cells exposed to high-LET (linear energy transfer) protons. The proton beams accelerated up to 45 MeV were horizontally extracted from the cyclotron, and were delivered to the cells acutely through a home made prototype over a range of physical depths (in the form of a variable water column). The biological systems used here were two in vitro cell lines, including human embryonic kidney cells (HEK 293), and human breast adenocarcinoma cell line (MCF-7). Cells were exposed to unmodulated proton beam radiation at a dose of 50 Gy similar to that used in therapy. Resazurin metabolism assay was investigated for measurement of cell response to irradiation as a simple and non-destructive assay. In the resazurin reduction test the non-fluorescent probe dye is reduced to pink and highly fluorescent resorufin. The dose-depth distribution of proton beam obtained based on the highly sensitive laser-induced fluorometric determination of resorufin was found to coincide well with the data collected using conventional film based dosimetry. The resazurin method yielded data comparable with the optical micrographs of the irradiated cells, showing the least cell survival at the measured Bragg-peak position of 10 mm. In addition, fused silica capillary was used as a sample container to increase the probability for irradiated laser beam to probe and excite resorufin in small sample volume of the capillary. The developed method has the potential to serve as a non-destructive, sample-thrifty, and time saving tool to realize more realistic, practical dose-depth distribution of proton beam compared to conventional in vitro cell viability assessment techniques.